Figure 1.
Inhibition of NADPH oxidase activity abolishes intracellular ROS generation on melanoma cells MV3.
(A) After adhesion, melanoma cells were incubated with or without DPI (10 µM) for different times (0.5–3 h) and ROS generation was evaluated by dihydrorhodamine-123 (DHR) assay followed by fluorescence microscopy analysis. (B) MV3 cells were incubated for 1 h with DPI (10 µM), PEG-SOD (25 U/mL), PEG-CAT (200 U/mL), or PEG-CAT and PEG-SOD (200 U/mL, 25 U/mL, respectively). Cellular ROS production was measured by intracellular oxidation of DHE to ethidium assessed by HPLC. (C–E) MV3 cells were incubated with or without DPI (10 µM). Intracellular ROS production was measured by intracellular oxidation of CM-H2DCFDA (C), DAF-AM (D) or HPF (E), as described in Material and Methods. Data are expressed as mean ± SD of three independent experiments. * p<0.05 vs. control;
Figure 2.
Inhibition of NADPH oxidase activity reduces survival of human melanoma cells MV3.
(A–B) MV3 cells (6×103) were incubated for 48 h with or without DPI (A; 0.1–10 µM) or apocynin (B; 1–10 µM), and MTT assay was performed as indicated in Material and Methods. (C) MV3 cells (6×103) were incubated for 48 h with or without DPI (10 µM), apocynin (10 µM) or Cycloheximide (5 µM). Subsequently, sulforhodamine-B assay was performed as described in Materials and Methods. Results are shown as percentage of control and expressed as mean ± SD of at least three independent experiments performed in quintuplicate. *p<0.05 vs. control.
Figure 3.
Human melanoma cells MV3 express NOX4, but not NOX5 or NOX2-related NADPH oxidase subunits.
Total RNAs were extracted from MV3 melanoma cells and from human polymorphonuclear neutrophils (A–B, PMN), human melanocytes (B, NGM) or from a human microvasculature endothelial cells linage (C, HMEC-1), used as positive controls. The expression levels of p47phox (A), NOX4 (B) or NOX5 (C) were analyzed by RT-PCR, using GAPDH expression as internal control as described in Materials and Methods. (D) NOX4, NOX2, p22phox, p40phox, p47phox, p67phox and βTubulin protein expression were detected by western blotting as described in Materials and Methods. Data are expressed as mean ± SD of three independent experiments and images are representative of three independent experiments with similar results.
Figure 4.
NADPH oxidase regulates cytoskeleton dynamics and focal adhesion sites.
MV3 adherent cells were incubated with DPI (10 µM) for 0.5, 2 and 4 hours and double-stained for FAK (FITC, green) and F-actin (TRITC-phalloidin, red). Co-localization was recognized as yellow dots, as indicated by white arrows and F-actin cortical distribution indicated by blue arrows. Images were obtained with a laser scanning confocal microscope (100X). Images are representative of three independent experiments with similar results.
Figure 5.
Inhibition of NADPH oxidase activity reduces FAKY397 phosphorylation and FAK-Src association on human melanoma cells.
(A) DPI (10 µM) was added to adherent cells (MV3) for 2 hours. Afterwards, total extracts obtained and the content of FAK and FAKY397 was assessed by immunoblotting. Blots were analyzed by densitometry, and phospho-FAKY397/total FAK ratio content is expressed as arbitrary units. (B) After adhesion MV3 cells were incubated in the presence or absence of DPI (10 µM) for 2 hours. Cells were then harvested, lysed and cell extracts were immunoprecipitated with anti-FAK and Western blots were performed for FAK and cSrc detection. Blots were analyzed by densitometry, and cSrc/total FAK ratio content was expressed as arbitrary units. (C) DPI (10 µM) was added to adherent cells (MV3) for 2 hours. Afterwards, total extracts obtained and the content of cSrc and βTubulin was assessed by immunoblotting. Blots were analyzed by densitometry, and cSrc/βTubulin ratio content is expressed as arbitrary units. *p<0.05 vs. control. Images are representative of three independent experiments.
Figure 6.
Inhibition of NADPH oxidase activity induces apoptosis on human melanoma cells.
(A) After adhesion, MV3 cells were incubated in the presence or absence DPI (10 µM) or CHX (5 µM) for 18 h. Cells were lysed and cell extracts submitted to SDS-PAGE in order to detect procaspase-3 and cleaved caspases-3 content, which were then analyzed by densitometry. ERK served as a loading control. Data shown are representative of three independent experiments with similar results. (B) After adhesion, cells were incubated in the absence or in the presence of DPI (10 µM) or CHX (5 µM) for 24 h. Cells were then harvested, fixed and stained with PI. Flow cytometric analysis of DNA contents was determined using WINMDI software. Percentage of apoptotic (in sub-G0) cells are expressed as mean ± SD of three independent experiments. *p<0.05 vs. control.
Figure 7.
NOX4 silencing reduces MV3 melanoma basal ROS production.
(A) Total mRNAs were extracted from the MV3 cells carrying scramble siRNA (Sc) or NOX4-specific siRNA (siRNA) and qRT-PCR was performed to analyze NOX4 mRNA expression, with actin as internal control as described in Materials and Methods. (B) Lysates obtained from transfected cells (106 cells) were subjected to immunoblotting to detect NOX4. Data are expressed as mean ± SD of three independent experiments and images are representative of three independent experiments with similar results. *p<0.05 vs. Scramble. (C) ROS detection assay (CM-H2DCFDA) was performed with MV3 melanoma cells transfected with Scramble or NOX4 siRNA, as described in Materials and Methods. Results are shown as percentage of Scramble of three independent experiments *p<0.05 vs. Scramble.
Figure 8.
NOX4 silencing reduces MV3 melanoma cell survival and FAK phosphorylation in MV3 melanoma cells.
Lysates obtained from transfected cells (106 cells) were subjected to immunoblotting to detect phospho-FAKY397 (A) and caspase-3 (C). (A) Data are expressed as mean ± SD of three independent experiments and images are representative of three independent experiments with similar results. *p<0.05 vs. Scramble. (C) Data shown are representative of three independent experiments with similar results. JC-1 (B), Sulforhodamine-B (D) and MTT (E) assays were performed with MV3 melanoma cells transfected with Sc or siRNA as described in Materials and Methods and the results are shown as percentage of Scramble of three independent experiments *p<0.05 vs. Scramble.
Figure 9.
A schematic model for signaling pathway activated by NADPH oxidase-generated ROS on human melanoma cells.
NADPH oxidase-derived ROS down-modulate protein tyrosine phosphatases, consequently increasing FAK phosphorylation. FAKY397 phosphorylation creates a high-affinity site for cSrc, as well as stimulates actin polymerization and focal adhesion stabilization, positively modulating cell motility, proliferation and survival. NADPH oxidase inhibition by DPI or NOX4 siRNA reduces ROS production, what probably leads to an increased protein tyrosine phosphatase activity, which, in turn, could inhibit focal adhesion formation/stabilization and induce MV3 human melanoma cells death.