Figure 6
NaV1.6 channels activity induces secretion of MMP-2 in cervical cancer cell lines. (A) Gelatin zymography for conditioned medium from cervical cancer cell lines. C33A, SiHa and HeLa cells were transfected with Nav1.6 and grown for 24 h in absence or presence of 1 µM TTX. Conditioned medium was obtained and cells were lysed. Activity for gelatinases MMP-2 and MMP-9 was analyzed on equal volumes of concentrated conditioned medium by using gelatin-substrate polyacrylamide gel electrophoresis followed by an incubation in activity buffer and a staining with Coomassie blue. The conditioned medium obtained from MCF-7 cells treated with 100 ng/ml phorbol 12, 13-dibutyrate (PDB) for 40 h, was used as positive control. Proteolytic activity was detected as clear bands against a dark background of undigested substrate (upper panel). Total protein extracts from cell lysates were analyzed by western blotting with anti-GAPDH antibody (bottom panel). The results shown are representative of three independent experiments. (B) Secretion of MMP-2 was quantified by densitometry analysis using GAPDH bands for normalizing. Results are given as the amount of gelatin degradation showed as clear bands relative to GAPDH bands for each condition. Columns are means ± SD from three independent experiments. *Statistically significant as P < 0.05. (C) Representative western blot for MMP-2 expression in total protein extracts from human biopsies of NCC and CeCa. Blots were stripped and re-probed for total GAPDH as the loading control. (D) Expression of MMP-2 protein was studied by densitometry analysis of western blot experiments. Results are given as the amount of MMP-2 protein relative to that of GAPDH for NCC (n = 13) and CeCa (n = 14). Columns are means ± SEM. * Statistically significant as P < 0.05. (E) Western blot analysis of NHE-1 expression and (F) relative levels of NHE-1 protein in the same samples of panel (C) and (D), respectively. Data are means ± SEM, *P < 0.05. Samples used in (C) and (E) are exactly the same.
