Journal: Scientific Reports

Article Title: The invasiveness of human cervical cancer associated to the function of Na V 1.6 channels is mediated by MMP-2 activity

doi: 10.1038/s41598-018-31364-y

Figure Lengend Snippet: Exploration of SCN8A expression in the neoplasia-carcinoma sequence of human cervical tissue. ( A ) Expression levels of SCN8A gene in low-grade (CIN1, n = 23) and high-grade cervical intraepithelial neoplasia (CIN2/3, n = 16), as well as in invasive cervical cancer positive to HPV16 (HPV16-CeCa, n = 35) and invasive cervical cancer positive to other oncogenic HPV types (HPV-CeCa, n = 22), versus non-cancerous cervix (NCC, n = 20). Bars show the average fold-change ratio (2 −ΔΔCt ) of SCN8A gene for individual samples of each group. ( B ) Scattering plot of fold-change values for each group of samples. Horizontal black lines represent the fold-change mean values. SCN8A gene was significantly upregulated in cervical cancer tissues (Mann-Whitney U test, P < 0.0001). ( C ) Western blot analysis of Na V 1.6 channel expression in total protein extracts from NCC, CIN and CeCa samples. Total protein extracts from HEK293 cells stably expressing Na V 1.6 channels were used as positive control. Representative results of four independent experiments. Numbers below blots correspond to sample number. Samples 266 and 275 are those that were present in all western blots experiments shown in the present work. ( D ) Relative expression of Na V 1.6 protein in cervical tissue samples. Blots were quantified by densitometry and normalized to that of GAPDH for NCC ( n = 12), CIN ( n = 10) and CeCa ( n = 29). Asterisks indicates P < 0.05 with a Student’s t-test.

Article Snippet: Endogenous peroxidase was then blocked by a commercial solution (Dako REAL, Dako) and incubated overnight at 4 °C with a rabbit-anti-Na V 1.6 primary antibody (1:150; Alomone).

Techniques: Expressing, Sequencing, MANN-WHITNEY, Western Blot, Stable Transfection, Positive Control

Journal: Scientific Reports

Article Title: The invasiveness of human cervical cancer associated to the function of Na V 1.6 channels is mediated by MMP-2 activity

doi: 10.1038/s41598-018-31364-y

Figure Lengend Snippet: Immunohistochemical analysis of Na V 1.6 channels in human cervical tissue. ( A ) Representative images of non-cancerous cervix (NCC), low-grade cervical intraepithelial neoplasia (CIN1), high-grade cervical intraepithelial neoplasia (CIN3) and invasive cervical cancer (CeCa), showing H&E staining (left column); immunohistochemical detection of Na V 1.6 channel at ×10 and ×40 amplifications (middle columns); and negative controls (right column), where a non-specific IgG primary antibody was used in parallel slides. Scale bars represent 100 μm, and 300 μm for ×40 amplifications, respectively. ( B ) Fraction of total staining for each sample analyzed. Immunoreactivity intensity was evaluated as no signal (NS, black), Low (orange) and high (red) Na V 1.6 staining in NCC, CIN1, CIN3 and CeCa tissue samples. The number of analyzed slides is indicated in parenthesis at the top of each column.

Article Snippet: Endogenous peroxidase was then blocked by a commercial solution (Dako REAL, Dako) and incubated overnight at 4 °C with a rabbit-anti-Na V 1.6 primary antibody (1:150; Alomone).

Techniques: Immunohistochemical staining, Staining

Journal: Scientific Reports

Article Title: The invasiveness of human cervical cancer associated to the function of Na V 1.6 channels is mediated by MMP-2 activity

doi: 10.1038/s41598-018-31364-y

Figure Lengend Snippet: Expression of Na V 1.6 channels in human cervical cancer cell lines. ( A ) Immunofluorescence confocal microscopy analysis of Na V 1.6 channels expression in C33A, SiHa, HeLa, and HEK293 cells stably expressing Na V 1.6 channels (HEK-Nav1.6; positive control). Cervical cancer cells were incubated with an antibody against Na V 1.6 protein followed by a staining with FITC-coupled secondary antibody. DAPI reagent was used for nucleus staining. Image acquisition was performed each 0.33 µm in a total thickness of 6.5 µm. Confocal sections were merged and 3D-reconstructions were performed from Z-planes for each region of interest. Orthogonal projections from xz and yz planes of confocal images show positive signal for Na V 1.6 protein ( far right panel ), indicated by red arrows in xz and yellow arrows in yz planes, respectively. Scale bar, 10 µm. ( B ) Western blot analysis of Na V 1.6 channel protein in total (T), cytoplasmic (C) and nuclear (N) protein extracts from human cervical cancer cell lines. Histone-3 and GAPDH proteins were used to demonstrate the enriching of subcellular fractions. Representative blot of three independent experiments. Notice that a ~150 kDa anti-Na V 1.6 reactive protein (red arrows) was found in nuclear fraction from cancer cells. The full-length ~250 kDa Na V 1.6 protein was only observed in total and cytoplasmic protein extract from C33A cells.

Article Snippet: Endogenous peroxidase was then blocked by a commercial solution (Dako REAL, Dako) and incubated overnight at 4 °C with a rabbit-anti-Na V 1.6 primary antibody (1:150; Alomone).

Techniques: Expressing, Immunofluorescence, Confocal Microscopy, Stable Transfection, Positive Control, Incubation, Staining, Western Blot

Journal: Scientific Reports

Article Title: The invasiveness of human cervical cancer associated to the function of Na V 1.6 channels is mediated by MMP-2 activity

doi: 10.1038/s41598-018-31364-y

Figure Lengend Snippet: The heterologous expression of Na V 1.6 channels boost the invasive capacity of cervical cancer cell lines. ( A ) Representative images of phase contrast and fluorescent microscopy of C33A cells 36 h after co-transfection with Na V 1.6 and GFP cDNAs. GFP-fluorescence indicated that 50–60% cancer cells were positively transfected. ( B ) Representative families of sodium currents obtained from non-transfected (black traces) and transfected C33A cells (red traces) with the Na V 1.6 channel in response to 16-ms pulses that depolarized the cell membrane from −80 to +80 mV in 10-mV steps applied every 10 s from a holding potential of −100 mV. Dotted lines indicate the baseline (zero current). Shown recordings are the average of two current traces at any given membrane potential and filtered at 5 kHz. ( C ) Current-voltage relationship for Na V 1.6 channels heterologously expressed in C33A cells. Peak Na + currents were averaged and plotted as a function of the depolarizing potential ( V m ). ( D ) Activation of normalized Na + conductance. Same cells as in ( C ). Smooth line is the fit to a Boltzmann function (see Methods) with the following parameters: V 1/2 = −12.2 ± 1.2 mV and k = 9.7 ± 1.0 mV; n = 9 cells. ( E ) The heterologous expression of Na V 1.6 channels enhances the invasive capacity of C33A cells. Relative invasion of C33A cells transfected with Na V 1.6 in absence or presence of 1 µM TTX, with respect to the control, untransfected C33A cells (black column). Columns represent the mean value of three independent experiments performed in triplicate (mean ± SD). *Statistically different from control condition ( P < 0.05).

Article Snippet: Endogenous peroxidase was then blocked by a commercial solution (Dako REAL, Dako) and incubated overnight at 4 °C with a rabbit-anti-Na V 1.6 primary antibody (1:150; Alomone).

Techniques: Expressing, Microscopy, Cotransfection, Fluorescence, Transfection, Activation Assay

Journal: Scientific Reports

Article Title: The invasiveness of human cervical cancer associated to the function of Na V 1.6 channels is mediated by MMP-2 activity

doi: 10.1038/s41598-018-31364-y

Figure Lengend Snippet: Na V 1.6 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 Na v 1.6 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.

Article Snippet: Endogenous peroxidase was then blocked by a commercial solution (Dako REAL, Dako) and incubated overnight at 4 °C with a rabbit-anti-Na V 1.6 primary antibody (1:150; Alomone).

Techniques: Activity Assay, Zymography, Transfection, Polyacrylamide Gel Electrophoresis, Incubation, Staining, Positive Control, Western Blot, Expressing

Journal: Scientific Reports

Article Title: The invasiveness of human cervical cancer associated to the function of Na V 1.6 channels is mediated by MMP-2 activity

doi: 10.1038/s41598-018-31364-y

Figure Lengend Snippet: The promotion of CeCa cell invasiveness by Na V 1.6 channels activity is mainly through secretion of pro- and mature MMP-2 forms. ( A ) Effect of protease inhibitors and EIPA on invasive capacity of Na V 1.6-transfected C33A cells. Cells C33A transfected with Na V 1.6 were seeded at cellular density of 6 × 10 4 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 Na V 1.6 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 Na V 1.6-transfected C33A cells (solid red columns).

Article Snippet: Endogenous peroxidase was then blocked by a commercial solution (Dako REAL, Dako) and incubated overnight at 4 °C with a rabbit-anti-Na V 1.6 primary antibody (1:150; Alomone).

Techniques: Activity Assay, Transfection, Staining, MANN-WHITNEY, Western Blot, Centrifugation, Positive Control, Expressing