Extracellular Superoxide Dismutase Regulates the Expression of Small GTPase Regulatory Proteins GEFs, GAPs, and GDI

<div><p>Extracellular superoxide dismutase (SOD3), which catalyzes the dismutation of superoxide anions to hydrogen peroxide at the cell membranes, regulates the cellular growth in a dose-dependent manner. This enzyme induces primary cell proliferation and immortalization at low expression levels whereas it activates cancer barrier signaling through the p53-p21 pathway at high expression levels, causing growth arrest, senescence, and apoptosis. Because previous reports suggested that the SOD3–induced reduction in the rates of cellular growth and migration also occurred in the absence of functional p53 signaling, in the current study we investigated the SOD3-induced growth-suppressive mechanisms in anaplastic thyroid cancer cells. Based on our data, the robust over-expression of SOD3 increased the level of phosphorylation of the EGFR, ERBB2, RYK, ALK, FLT3, and EPHA10 receptor tyrosine kinases with the consequent downstream activation of the SRC, FYN, YES, HCK, and LYN kinases. However, pull-down experiments focusing on the small GTPase RAS, RAC, CDC42, and RHO revealed a reduced level of growth and migration signal transduction, such as the lack of stimulation of the mitogen pathway, in the SOD3 over-expressing cells, which was confirmed by MEK1/2 and ERK1/2 Western blotting analysis. Interestingly, the mRNA expression analyses indicated that SOD3 regulated the expression of guanine nucleotide-exchange factors (<i>RHO GEF16</i>, <i>RAL GEF RGL1</i>), GTPase-activating proteins (<i>ARFGAP ADAP2</i>, <i>RAS GAP RASAL1</i>, <i>RGS4</i>), and a Rho guanine nucleotide-disassociation inhibitor (<i>RHO GDI 2</i>) in a dose dependent manner, thus controlling signaling through the small G protein GTPases. Therefore, our current data may suggest the occurrence of dose-dependent SOD3–driven control of the GTP loading of small G proteins indicating a novel growth regulatory mechanism of this enzyme.</p></div

Robust SOD3 expression reduced anaplastic thyroid cancer cell growth.

<p>(a) Real time RT-PCR analysis showed a high-level mRNA production of <i>SOD3</i> transgene in anaplastic thyroid cancer 8505c cells. (b,c) Western blotting and related band intensity analysis showed high-level SOD3 production caused by <i>SOD3</i> transgene. (d) The growth curve analysis suggested significantly (p<0.001) decreased cell numbers in cells transfected with <i>SOD3</i>. (e,f) The BrdU DNA incorporation analysis confirmed significantly (p<0.001) decreased cellular growth caused by SOD3. (g) The 3-D gel invasion assay suggested almost complete lack of invasive capacity of 8505c SOD3 cells. (h,i) The soft agar analysis showed significantly (p<0.001) reduced formation of colonies suggesting decreased anchorage independent growth ability for SOD3 cells as compared to 8505c control vector transfected cells. (j) The growth curve analysis of control and SOD3 cells in the presence of 2.5 mM N-Acetyl-cysteine (NAC) showed growth reduction both in 8505c control and SOD3 expressing cells upon the treatment. (k) <i>CATALASE</i> mRNA expression. The quantitative RT-PCR showed no difference between 8505c control and SOD3 cells in <i>CATALASE</i> expression. Data are expressed as mean ±SD. The p-values (* = p<0.05, ** = p<0.01, *** = p<0.001).</p

Growth-associated genes showing SOD3-mediated downregulated expression.

<p>Growth-associated genes showing SOD3-mediated downregulated expression.</p

SOD3 regulated the expression of small GTPase regulatory genes and affected the activation of small GTPases.

<p>(a-f) Quantitative RT-PCR for GTPase superfamily regulatory genes. The panels show the expression levels of the regulatory genes <i>in vitro</i> cultured cells containing high-level <i>SOD3</i> mRNA and reduced growth abilities. (g-k) Pull-down assay for small GTPase proteins RAS, RAC, CDC42, and RHO. The assay suggested significantly (p<0.001) reduced GTP loading to each tested GTPase therefore being in line with reduced cell proliferation, migration, and growth related signal transduction. Data are expressed as mean ±SD. The p-values (* = p<0.05, ** = p<0.01, *** = p<0.001).</p

Reversed growth characteristics by <i>H-RasV12</i> and <i>RHO GEF16</i> over-expression in <i>SOD3</i> transfected cells.

<p>(a,b) The 8505c SOD3 cells were stable transfected with <i>H-RasV12</i> oncogene and analyzed by growth curve and 3-D invasion analysis. Both analysis suggested marked increase in the growth of SOD3 cells after <i>H-RasV12</i> transfection. (c) The 8505c SOD3 cells were stably transfected with <i>RHO GEF16</i>. The real time RT-PCR suggested significant (p<0.01) increase in <i>RHO GEF16</i> mRNA production that was confirmed by Western blotting (lower panel). (d,e) The BrdU DNA incorporation analysis showed significantly (p<0.001) increased nuclear proliferation after <i>RHO GEF16</i> over-expression as compared to 8505c SOD3 cells. (f) The RNAi of <i>RGS4</i> in 8505c SOD3 cells showed significant downregulation of <i>RGS4</i> mRNA production. (g,h) However, the BrdU DNA incorporation analysis failed to show differences between 8505c control and 8505c SOD3/siRGS4 cells. (i) A schematic drawing hypothesizing the SOD3 action in the cells. SOD3 over-expression activates cell membrane RTKs and membrane associated non-receptor tyrosine kinases. Depending on the expression level of <i>SOD3</i> the proliferative and migratory signal is either halted or promoted at the level of small GTPase regulatory genes and small GTPases.</p

Western blotting and DNA damage response analysis.

<p>(a,b) Western blotting analyses supported the protein array data showing the increased activation of RTKs and non-receptor kinases in the <i>SOD3</i>-transfected 8505c cells. Note, MEK1/2 and ERK1/2 phosphorylation was moderately downregulated based on the band density analyses. (c,d) Western blotting and the related band intensity analysis for β-catenin nuclear and cytosolic proteins showed significantly (p<0.001) increased β-catenin production in cytosolic compartment. Tubulin was used to normalize cytosolic proteins and SP1 was used to normalize nuclear proteins. (e,f) γH2AX staining for 8505c cells suggested significantly (p<0.001) increased histone H2AX phosphorylation, suggesting DNA damages in 8505c SOD3 cells, which supported the increased CHK2 phosphorylation observed in protoarray and in the Western blotting. The white arrows in the control and the SOD3 merge panels indicate cells that are magnified to detect the nuclear foci. The white arrows in the control and the SOD3 magnification images indicate the nuclear foci. Data are expressed as mean ±SD. The p-values (* = p<0.05, ** = p<0.01, *** = p<0.001).</p

MOESM1 of Clinical relevance of thyroid cell models in redox research

Additional file 1: Table S1. PCR primers