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Superoxide dismutase 1 (SOD1) is essential for H2O2-mediated oxidation and inactivation of phosphatases in growth factor signaling - PubMed

  • ️Tue Jan 01 2008

Superoxide dismutase 1 (SOD1) is essential for H2O2-mediated oxidation and inactivation of phosphatases in growth factor signaling

Jose C Juarez et al. Proc Natl Acad Sci U S A. 2008.

Abstract

Superoxide dismutase 1 (SOD1) is an abundant copper/zinc enzyme found in the cytoplasm that converts superoxide into hydrogen peroxide and molecular oxygen. Tetrathiomolybdate (ATN-224) has been recently identified as an inhibitor of SOD1 that attenuates FGF-2- and VEGF-mediated phosphorylation of ERK1/2 in endothelial cells. However, the mechanism for this inhibition was not elucidated. Growth factor (GF) signaling elicits an increase in reactive oxygen species (ROS), which inactivates protein tyrosine phosphatases (PTP) by oxidizing an essential cysteine residue in the active site. ATN-224-mediated inhibition of SOD1 in tumor and endothelial cells prevents the formation of sufficiently high levels of H(2)O(2), resulting in the protection of PTPs from H(2)O(2)-mediated oxidation. This, in turn, leads to the inhibition of EGF-, IGF-1-, and FGF-2-mediated phosphorylation of ERK1/2. Pretreatment with exogenous H(2)O(2) or with the phosphatase inhibitor vanadate abrogates the inhibition of ERK1/2 phosphorylation induced by ATN-224 or SOD1 siRNA treatments. Furthermore, ATN-224-mediated SOD1 inhibition causes the down-regulation of the PDGF receptor. SOD1 inhibition also increases the steady-state levels of superoxide, which induces protein oxidation in A431 cells but, surprisingly, does not oxidize phosphatases. Thus, SOD1 inhibition in A431 tumor cells results in both prooxidant effects caused by the increase in the levels of superoxide and antioxidant effects caused by lowering the levels of H(2)O(2). These results identify SOD1 as a master regulator of GF signaling and as a therapeutic target for the inhibition of angiogenesis and tumor growth.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

SOD1 is the target for ATN-224 and SOD1 inhibition attenuated the phosphorylation of ERK1/2 upon GF stimulation. (A) A431 cells were treated with ATN-224 for 48 h, and proliferation (MTT assay) (inverted filled triangles) and SOD1 activity (filled circles) were determined. (B) A431 cells were transfected with SOD1 siRNA (74) (filled circles), control siRNA (inverted filled triangles), or nontransfected (open circles) and plated on a 96-well plate, and proliferation was determined as in A. (C) A431 proliferation (MTT assay) was measured in cells treated with MnTBAP either alone (filled bars) or with 30 μM ATN-224 (empty bars). (D) A431 or A549 cells were treated with ATN-224 for 48 h. Then, cells were stimulated with 10 ng/ml of EGF for 10 min and lysed, and Western blot analyses were carried out. (E) HT-29 cells were incubated with ATN-224 for 48 h. Then, cells were stimulated with IGF-1 for 10 min and lysed, and ELISAs were carried out. (F) A431 cells were transfected with different SOD1 siRNA [74, 75, 76, and all three (comb.)] or control siRNA (C) for 48 h. Then, the cells were stimulated with 10 ng/ml of EGF for 10 min and lysed, and Western blot analyses were carried out.

Fig. 2.
Fig. 2.

SOD1 inhibition attenuates GF receptor phosphorylation. (A) A431 cells were incubated with the indicated amounts of ATN-224 for 48 h. Then, cells were stimulated with 100 ng/ml of EGF for 10 min and lysed and Western blot analyses were carried out, probing for EGFR and pEGFR (Tyr-1173). (B) A431 cells were incubated with ATN-224 for 48 h. Then, cells were stimulated with 100 ng/ml of EGF for 10 min and lysed and Western blot analyses were carried out, probing for pEGFR (Tyr-845, -992, -1045, -1068, -1086, -1148, and -1173) and tubulin. The appropriate bands were quantitated and normalized to tubulin. (C) HT-29 cells were incubated with ATN-224 for 48 h. Then, cells were stimulated with 20 ng/ml of IGF-1 and lysed, and Western blot analyses were carried out, probing for IGF-1Rβ or pIGF-1Rβ (Tyr-1135/1136). (D) U87 cells were incubated with ATN-224 for 48 h in full growth media. Then, cells were lysed, and Western blot analyses were carried out.

Fig. 3.
Fig. 3.

ATN-224 treatment of A431 cells alters the redox state, which seems responsible for the inhibition of ERK1/2 phosphorylation. (A) A431 cells were incubated with ATN-224 for 48 h in full growth media. Then, cells were treated with dihydroethidium (DHE) at 1.6 μM in PBS for 15 min followed by extensive washing and analyzed by flow cytometry to determine the levels of superoxide (n = 3; *, P < 0.001). (B) A431 cells were incubated with ATN-224 for 48 h in full growth media. Then, cytosolic aconitase activity was measured (average of n = 3 ± standard deviation) as described in Materials and Methods. (C) A431 cells were incubated with 7 μM ATN-224 for 48 h in full growth media. Then, the cells were treated with 1, 10, or 100 μM of H2O2 for 0.5 h in cell media containing 0.5% FBS, washed, and then stimulated with 10 ng/ml of EGF. The numbers under the bands [relative intensity (RI)] represent the intensity of the pERK bands normalized to tubulin. (D) A431 cells were treated with ATN-224 for 24 or 48 h, cells were lysed, and Western blot analyses were carried out, probing with antibodies against Prx II and Prx-SO3. The later recognizes the oxidized forms of Prx I to IV. (E) A431 were incubated with ATN-224 for 48 h in full growth media. Then, cells were treated with H2O2 in cell media containing 0.5% FBS for 10 min. After that, cells were washed, lysed in the presence of NEM, run on a nonreducing SDS/PAGE, transferred, and probed with antibodies against PTEN. When PTEN is oxidized by H2O2, it runs as a faster species than reduced PTEN in a SDS/PAGE. The numbers under the bands (RI) represent the intensity of reduced PTEN bands normalized to tubulin.

Fig. 4.
Fig. 4.

Inhibition of SOD1 by ATN-224 reduces the levels of oxidized PTP1B and other proteins. (A) A431 cells treated with 5 μM ATN-224 for 48 h were lysed in the presence of iodoacetic acid to block reduced Cys residues, buffer exchanged, reduced with DTT and reacted with maleimide-biotin so that originally oxidized proteins would acquire a biotin label. Proteins were then IP with streptavidin–agarose (SA) followed by Western blot analysis, which was probed for biotin. (B) A431 cells were treated with 5 μM ATN-224 for 48 h and then stimulated with 100 ng/ml of EGF for 5 min and treated as in B, and biotinylated proteins were then immunoprecipitated with SA followed by Western blot analysis, which was probed for PTP1B.

Fig. 5.
Fig. 5.

PTP inhibitors block ATN-224 and SOD1 siRNA-mediated attenuation of ERK1/2 phosphorylation. (A) HUVEC were incubated with ATN-224 for 48 h in full growth media. Cells were then treated with orthovanadate for either 15 or 30 min followed by stimulation with FGF-2 and lysed, and Western blot analyses were carried out. The numbers under the bands (relative intensity) represent the intensity of the pERK bands normalized to tubulin. (B) A431 cells were treated either with 7 μM ATN-224 (224) for 48 h or transfected with SOD1 siRNA or control siRNA for 48 h. Cells were then treated with 1 mM orthovanadate (V) for 15 min or not treated before stimulation with 10 ng/ml of EGF for 10 min. Cells were lysed, and Western blot analyses were carried out. V, orthovanadate treatment; 224, ATN-224 treatment. (C) A431 cells were treated with ATN-224 for 48 h and exposed to a PTP1B inhibitor 1 h before being stimulated with 10 ng/ml of EGF for 10 min. Cells were lysed, and ELISAs were carried out, probing for ERK1/2, pERK. The graph shows means ± SD (n = 3) of pERK normalized to ERK1/2.

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