Superoxide mediates direct current electric field-induced directional migration of glioma cells through the activation of AKT and ERK.

Direct current electric fields (DCEFs) can induce directional migration for many cell types through activation of intracellular signaling pathways. However, the mechanisms that bridge extracellular electrical stimulation with intracellular signaling remain largely unknown. In the current study, we found that a DCEF can induce the directional migration of U87, C6 and U251 glioma cells to the cathode and stimulate the production of hydrogen peroxide and superoxide. Subsequent studies demonstrated that the electrotaxis of glioma cells were abolished by the superoxide inhibitor N-acetyl-l-cysteine (NAC) or overexpression of mitochondrial superoxide dismutase (MnSOD), but was not affected by inhibition of hydrogen peroxide through the overexpression of catalase. Furthermore, we found that the presence of NAC, as well as the overexpression of MnSOD, could almost completely abolish the activation of Akt, extracellular-signal-regulated kinase (Erk)1/2, c-Jun N-terminal kinase (JNK), and p38, although only JNK and p38 were affected by overexpression of catalase. The presenting of specific inhibitors can decrease the activation of Erk1/2 or Akt as well as the directional migration of glioma cells. Collectively, our data demonstrate that superoxide may play a critical role in DCEF-induced directional migration of glioma cells through the regulation of Akt and Erk1/2 activation. This study provides novel evidence that the superoxide is at least one of the "bridges" coupling the extracellular electric stimulation to the intracellular signals during DCEF-mediated cell directional migration

Superoxide-mediated electrotaxis depends on the activation of Akt and Erk1/2.

<p>DCEF would induce the phosphorylation of Erk1/2 and Akt, and the activation of Erk1/2 or Akt were reduced by 20 µM PD98059 (inhibitor of Erk) or 20 µM LY294002 (inhibitor of Akt) respectively (A). DCEF-induced directional migration of U87 cells was also significantly decreased by PD98059 (B) or LY294002 (C). Bar = 25 µm.</p

NAC blocks DCEF-induced directional migration of glioma cells.

<p>U87 (A and A′), C6 (B and B′) and U251 (C and C′) glioma cells migrate in all directions after two hours of NAC (10 mM) pretreatment with or without stimulation by a 200 mV/mm DCEF. ROS generation in cells stimulated with a 200 mV/mm DCEF for 30 minites with or without pretreatment with NAC (10 mM) as detected by DCFH-DA (D). Superoxide was detected by HE (E). Analysis of direction (cosθ) of U87 glioma cell migration (F). Data are expressed as the mean ± standard deviation (SD) (n = 3). * Compared with the NAC-pretreated cells, <i>P</i><0.05.</p

DCEF-stimulated activation of Erk1/2, JNK, and p38 in U87 glioma cells.

<p>U87 glioma cells were stimulated with 200 mV/mm DCEF for 0, 15, 30, 45, or 60 minutes (A). The cell lysate was probed with antibodies specific for p-Akt, total Akt, p-Erk1/2, total Erk1/2, p-JNK, total JNK, p-p38, and total p38. Activation of Erk1/2, JNK, and p38 in U87 glioma cells with or without 10 mM NAC pretreatment were stimulated with 200 mV/mm DCEF for 0, 30, and 60 minutes (B). Activation of Erk1/2, JNK, and p38 in U87 glioma cells with or without overexpression of mCat were stimulated with 200 mV/mm DCEF for 0, 30, and 60 minutes (C). Activation of Erk1/2, JNK, and p38 in U87 glioma cells with or without overexpression of MnSOD were stimulated with 200 mV/mm DCEF for 0, 30, and 60 minutes (D). Data presented are a typical representation of a duplicated experiment.</p

Schematic diagram of superoxide acting as a “bridge” between the extracellular electric stimulation and the intracellular signals in electrotaxis.

<p>DCEF induces the generation of superoxide. The superoxide then activates the Erk1/2 and Akt. The activation of these signaling pathways might be critical for the rearrangement and polarization of the cytoskeleton, which gears up the directional migration.</p

DCEF-mediated induction of directional migration of glioma cells.

<p>In the absence of DCEF, Astrocytes (A), U87 glioma cells (B), C6 glioma cells (C) and U251 glioma cells (D) migrated with equal probability in all directions. The DCEF in a 200 mV/mm could not direct the migration of the astrocyte (A′). Glioma cells migrated toward the cathode in a 200 mV/mm DCEF after two hours of exposure (B′–D′). An analysis of direction (cosθ) and speed (µm/h) of glioma cells migration (E and F). *<i>P</i><0.05.</p

Phosphorylation levels of 60 selected proteins related to cell migration were detected by a Full Moon Cytoskeleton II Phosphorylation Antibody Array.

<p>The results showed the folds of phosphorylation levels 30 min after the introduction of 200mV/mm DCEF than that without DCEF.</p

DCEF induces the generation of ROS in U87, C6 and U251 glioma cells.

<p>Generation of intracellular ROS was detected by the semi-quantitative technique based on the use of DCFH-DA (A) and HE (B) 15, 30, and 60 minutes after the treatment of cells with 200 mV/mm. Cells without DCEF stimulation were used as the negative control. Generation of intracellular hydrogen peroxide was detected by DCFH-DA (green fluorescence) and superoxide was detected by HE (red fluorescence) 30 minutes after EF treatment with a strength of 200 mV/mm (C). *Compared with the negative control, <i>P</i><0.05; Bar = 50 µm.</p