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, 7 (1), 4814

Polysulfide Na 2 S 4 Regulates the Activation of PTEN/Akt/CREB Signaling and Cytotoxicity Mediated by 1,4-naphthoquinone Through Formation of Sulfur Adducts

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Polysulfide Na 2 S 4 Regulates the Activation of PTEN/Akt/CREB Signaling and Cytotoxicity Mediated by 1,4-naphthoquinone Through Formation of Sulfur Adducts

Yumi Abiko et al. Sci Rep.

Abstract

Electrophiles can activate redox signal transduction pathways, through actions of effector molecules (e.g., kinases and transcription factors) and sensor proteins with low pKa thiols that are covalently modified. In this study, we investigated whether 1,4-naphthoquinone (1,4-NQ) could affect the phosphatase and tensin homolog (PTEN)-Akt signaling pathway and persulfides/polysulfides could modulate this adaptive response. Simultaneous exposure of primary mouse hepatocytes to Na2S4 and 1,4-NQ markedly decreased 1,4-NQ-mediated cell death and S-arylation of cellular proteins. Modification of cellular PTEN during exposure to 1,4-NQ was also blocked in the presence of Na2S4. 1,4-NQ, at up to 10 µM, increased phosphorylation of Akt and cAMP response element binding protein (CREB). However, at higher concentrations, 1,4-NQ inhibited phosphorylation of both proteins. These bell-shaped dose curves for Akt and CREB activation were right-shifted in cells treated with both 1,4-NQ and Na2S4. Incubation of 1,4-NQ with Na2S4 resulted in formation of 1,4-NQ-S-1,4-NQ-OH. Unlike 1,4-NQ, authentic 1,4-NQ-S-1,4-NQ-OH adduct had no cytotoxicity, covalent binding capability nor ability to activate PTEN-Akt signaling in cells. Our results suggested that polysulfides, such as Na2S4, can increase the threshold of 1,4-NQ for activating PTEN-Akt signaling and cytotoxicity by capturing this electrophile to form its sulfur adducts.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Cytotoxicity and chemical modification of cellular proteins during treatment of cells with 1,4-NQ, with or without Na2S4. (A,B) Primary mouse hepatocytes were exposed to 1,4-NQ with or without 10 (A) or 100 (B) µM Na2S4 for 24 h. Cell viability was assessed with the MTT assay. Each value is the mean ± standard error for three independent experiments. *P < 0.05 and **P < 0.01, compared with the control. C, D: Primary mouse hepatocytes were exposed to 1,4-NQ, with or without 10 (C) or 100 (D) µM Na2S4 for 1 h. Covalent modification of cellular proteins by 1,4-NQ was detected by western blotting using an anti-1,4-NQ antibody (upper), proteins were detected following SDS-PAGE by Coomassie Brilliant Blue staining (middle) and intensities of modified protein bands were calculated with Multi Gauge software (lower). Representative data are shown from two independent experiments.
Figure 2
Figure 2
Chemical modification of cellular and recombinant PTEN by 1,4-NQ and suppression of 1,4-NQ modification of cellular PTEN. (A) Primary mouse hepatocytes were simultaneously treated with DMSO or 1,4-NQ and 10 (upper) or 100 (lower) µM Na2S4 for 30 min, then PTEN was immunoprecipitated using anti-PTEN antibody. Western blotting was performed using the indicated antibodies. Representative blots are shown from three independent experiments. (B) Recombinant GST-tagged human PTEN (1 μg) was incubated with 1,4-NQ (1–8 μM) at 25 °C for 1 h. The reaction mixture was then subjected to immunoblotting, with the anti-1,4-NQ antibody, and SDS-PAGE, with Coomassie Brilliant Blue staining. Representative blots are shown from three independent experiments. (C) Results of nanoUPLC-MSE analysis of 1,4-NQ-modified cysteine residues in GST-tagged human PTEN. Recombinant GST-tagged human PTEN (1.7 μg) was incubated with 1,4-NQ (10 μM) at 25 °C for 30 min in a total volume of 10 μL 50 mM Tris-HCl (pH 7.5). After the reaction, PTEN protein was digested with trypsin and analyzed by nanoUPLC-MSE. The corresponding MSE data are shown in Table 1.
Figure 3
Figure 3
Participation of polysulfide in the 1,4-NQ-mediated phosphorylation of Akt and CREB in primary mouse hepatocytes. (A) Cells were exposed to the indicated concentrations of 1,4-NQ for 30 min in the presence of Na2S4 at 0, 10 (A), or 100 (B) µM. Akt and CREB phosphorylation was determined by western blotting (right). Representative blots are shown from three independent experiments. Band intensities were normalized to those of total Akt and CREB, respectively (left). Intensities are presented as fold induced relative to results with 0 µM 1,4-NQ exposure. Each value is the mean ± standard error for three independent experiments. *P < 0.05 and **P < 0.01, compared with 0 µM 1,4-NQ exposure.
Figure 4
Figure 4
Purification and identification of the sulfur adduct of 1,4-NQ. (A) Separation of sulfur adducts of 1,4-NQ by column chromatography. 1,4-NQ (5 mM) was incubated with Na2S4 (10 mM) for 10 min at room temperature. The resulting solution was applied to an ODS column and eluted with 20% acetonitrile for 40 min followed by 80% acetonitrile for 60 min at a flow rate of 10 mL/min. Each fraction was analyzed by UV absorbance at 250 nm and by UPLC-MS. Fraction II primarily contained m/z 361 in negative ion mode. (B) FT-ICR-MS of the purified sulfur adduct. ESI-MS spectrum of the reaction product with m/z 361 (upper) and comparison of isotope ratios between the product and an elemental composition of C20H10O5S (lower). (C) Magnified views of the 1H NMR (upper) and 1H-1H COSY NMR (lower) spectra of a sulfur adduct of 1,4-NQ with m/z 361. Four doublet proton signals at δ 8.05 (d, J = 3.7 Hz, 1 H), 7.97 (d, J = 3.6 Hz, 1 H), 7.93 (d, J = 3.7 Hz, 1 H) and 7.91 (d, J = 5.8 Hz, 1 H), and four triplet proton signals at δ 7.86 (t, J = 7.4 Hz, 1 H), 7.83 (t, J = 7.4 Hz, 1 H), 7.74 (t, J = 7.5 Hz, 1 H) and 7.63 (t, J = 7.5 Hz, 1 H) were detected. An additional singlet proton signal in the high field at δ 6.07 (s, 1 H) must be attributable to H-3. An aromatic OH group must be located at the C-3′ position, although this OH signal was not detected. The COSY NMR spectrum showed that two triplets at δ 7.74 and δ 7.63 ppm were correlated to one another and to two doublets at δ 7.97 and δ 7.9 ppm, respectively. These signals must be attributable to H-5′, H-6′, H-7′ and H-8′. The other two triplets at δ 7.86 and δ 7.83 ppm were correlated to one another and to two doublets at δ7.93 and δ 8.05 ppm, respectively. These signals must be attributable to H-5, H-6, H-7 and H-8. D: MS spectrum of the sulfur adduct (m/z 361) of 1,4-NQ formed during incubation with Na2S4. The purified sulfur adduct was analyzed by UPLC-MS. Representative data are shown from three independent experiments.
Figure 5
Figure 5
Cytotoxicity, covalent protein modification and Akt–CREB signaling activation caused by exposure of primary mouse hepatocytes to 1,4-NQ-sulfur adducts. (A) Primary mouse hepatocytes were exposed to 1,4-NQ or 1,4-NQ–S–1,4-NQ-OH for 24 h. Cell viability was determined with the MTT assay. Each value is the mean ± standard error for three independent experiments. (B) Cells were exposed to 1,4-NQ–S–1,4-NQ-OH (20 µM) or 1,4-NQ (40 µM) for 1 h. Covalent modification of cellular proteins with 1,4-NQ, as detected by western blotting (left), BPM assay results (middle) and protein bands on SDS-PAGE, as stained by Coomassie Brilliant Blue (right). Representative blots are shown from three independent experiments. (C) Recombinant GST-tagged human PTEN (1 μg) was incubated with 1,4-NQ–S–1,4-NQ-OH (5 µM) or 1,4-NQ (10 µM) at 25 °C for 1 h and then further incubated with BPM (15 μM) at 37 °C for 30 min. The reaction mixture was then analyzed by immunoblotting with the anti-biotin antibody and Coomassie Brilliant Blue was used to visualize bands after SDS-PAGE. Representative blots are shown from three independent experiments. (D) Cells were exposed to different concentrations of 1,4-NQ-sulfide for 30 min. Akt and CREB phosphorylation was determined by western blotting (upper). Representative blots are shown from three independent experiments. Band intensities were normalized to those for total Akt and CREB, respectively (lower). Intensities are expressed as fold induced, relative to results with 0 µM 1,4-NQ exposure. Each value is the mean ± the standard error for three independent experiments. *P < 0.05 and *P < 0.01, compared with the 0 µM 1,4-NQ exposure.
Figure 6
Figure 6
1,4-NQ-mediated activation of PTEN–Akt signaling, which was suppressed by Na2S4.

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