Figure 7
The promotion of CeCa cell invasiveness by NaV1.6 channels activity is mainly through secretion of pro- and mature MMP-2 forms. (A) Effect of protease inhibitors and EIPA on invasive capacity of NaV1.6-transfected C33A cells. Cells C33A transfected with NaV1.6 were seeded at cellular density of 6 × 104 cells per insert in the absence (Control) or the presence of protease inhibitors (GM6001, 25 µM; E-64, 100 µM; Leupeptin, 100 µM), or the NHE-1 specific inhibitor (EIPA, 1 µM) using a serum gradient of 10% for 48 h. For these experiments, invasive cells were stained with DAPI, photographed and counted automatically. Results from six experimental observations of two independent experiments are expressed as relative invasion (mean ± SD), normalized to the control condition. Statistical difference at P < 0.05 for * and P < 0.01 for ** (Mann-Whitney Rank Sum test). (B) Representative western blotting experiment for the analysis of pro- and mature MMP-2 forms in supernatants of C33A cultures. Conditioned medium from C33A cells transfected with NaV1.6 and grown in the absence or the presence of 1 μM TTX was recovered after 48 h and concentrated by centrifugation. HSC70 was used as a loading control. Supernatants form C33A cells grown in complete medium (10% FBS) served as a positive control (+). (C) Evaluation of pro- and mature MMP-2 expression. Quantification was made by densitometric analysis of western blot images. Results are given as the amount of pro-MMP2 and MMP-2 protein relative to that of HSC70 in cell lysates. Columns are mean ± SD from three independent experiments. *Significantly different from Control at P < 0.01. There were no significant differences between proMMP-2 and MMP-2 forms in NaV1.6-transfected C33A cells (solid red columns).
