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. 2019 Sep:26:101290.
doi: 10.1016/j.redox.2019.101290. Epub 2019 Aug 2.

Redox modifications of cysteine-containing proteins, cell cycle arrest and translation inhibition: Involvement in vitamin C-induced breast cancer cell death

Nadine El Banna  1 , Elie Hatem  1 , Amélie Heneman-Masurel  1 , Thibaut Léger  2 , Dorothée Baïlle  1 , Laurence Vernis  1 , Camille Garcia  2 , Sylvain Martineau  1 , Corinne Dupuy  3 , Stéphan Vagner  1 , Jean-Michel Camadro  2 , Meng-Er Huang  4
Affiliations

Redox modifications of cysteine-containing proteins, cell cycle arrest and translation inhibition: Involvement in vitamin C-induced breast cancer cell death

Nadine El Banna et al. Redox Biol. 2019 Sep.

Abstract

Vitamin C (VitC) possesses pro-oxidant properties at high pharmacologic concentrations which favor repurposing VitC as an anti-cancer therapeutic agent. However, redox-based anticancer properties of VitC are yet partially understood. We examined the difference between the reduced and oxidized forms of VitC, ascorbic acid (AA) and dehydroascorbic acid (DHA), in terms of cytotoxicity and redox mechanisms toward breast cancer cells. Our data showed that AA displayed higher cytotoxicity towards triple-negative breast cancer (TNBC) cell lines in vitro than DHA. AA exhibited a similar cytotoxicity on non-TNBC cells, while only a minor detrimental effect on noncancerous cells. Using MDA-MB-231, a representative TNBC cell line, we observed that AA- and DHA-induced cytotoxicity were linked to cellular redox-state alterations. Hydrogen peroxide (H2O2) accumulation in the extracellular medium and in different intracellular compartments, and to a lesser degree, intracellular glutathione oxidation, played a key role in AA-induced cytotoxicity. In contrast, DHA affected glutathione oxidation and had less cytotoxicity. A "redoxome" approach revealed that AA treatment altered the redox state of key antioxidants and a number of cysteine-containing proteins including many nucleic acid binding proteins and proteins involved in RNA and DNA metabolisms and in energetic processes. We showed that cell cycle arrest and translation inhibition were associated with AA-induced cytotoxicity. Finally, bioinformatics analysis and biological experiments identified that peroxiredoxin 1 (PRDX1) expression levels correlated with AA differential cytotoxicity in breast cancer cells, suggesting a potential predictive value of PRDX1. This study provides insight into the redox-based mechanisms of VitC anticancer activity, indicating that pharmacologic doses of VitC and VitC-based rational drug combinations could be novel therapeutic opportunities for triple-negative breast cancer.

Keywords: Ascorbic acid; Breast cancer; Dehydroascorbic acid; Oxidative stress; Redoxome; Vitamin C.

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Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Sensitivity of TNBC, non-TNBC and noncancerous cells toward AA and DHA. (A-B) Seven TNBC cell lines were treated with AA (A) or DHA (B) ranging from 0 to 10 mM for 24 h. Cell viability (%) relative to non-treated condition was measured with MTT assay for each cell line. Data are means ± SD of at least 6 independent experiments. Statistical significance is assessed by two-way ANOVA with Sidak's multiple comparisons test. (C) MDA-MB-231 cells were treated with AA or DHA for 24 h. Cell viability (%) relative to non-treated condition was measured by MTT assay (left panel). Data are means ± SD of at least 6 independent experiments. Representative images of colonies formation of MDA-MB-231 cells after AA or DHA treatments for 24 h are shown (right panel). (D) Six non-TNBC cells were treated with AA ranging from 0 to 10 mM for 24 h. Cell viability (%) relative to non-treated condition was measured with MTT assay. Data are means ± SD of at least 6 independent experiments. Statistical significance is assessed by two-way ANOVA with Sidak's multiple comparisons test. (E) HMEC, normal human dermal fibroblasts and HUVEC were treated with AA ranging from 0 to 10 mM for 24 h. Cell viability (%) relative to non-treated condition was measured with MTT assay for each cell line. Data are means ± SD of 4 independent experiments. Statistical significance is assessed by ordinary two-way ANOVA with Tukey's multiple comparisons test. ****, P < 0.0001.
Fig. 2
Fig. 2
Glutathione oxidation and ROS generation in response to AA and DHA treatments in MDA-MB-231 cells. (A) GSH and GSSG quantities were assessed in non-treated cells and cells treated with 2.5 or 10 mM AA or DHA for ½ hour and 2 h. Treatment with 250 μM diamide for 10 min was used as a positive control (PC). GSH/GSSG ratio value in non-treated cells is set as 1 and relative GSH/GSSG ratios calculated in treated conditions are represented as means ± SD of 4 independent experiments. Statistical significance is assessed by ordinary one-way ANOVA with Tukey's multiple comparisons test. (B) General intracellular ROS was assessed by flow cytometry using carboxy-H2DCFDA probes in non-treated cells (NT), cells treated with 2.5 or 10 mM AA or DHA for 4 h (upper panel), cells treated with 10 mM AA, 10 mM AA plus 2000 U/ml catalase or 10 mM AA plus 10 mM GSH (lower panel). Treatment with 100 μM  H2O2 for 20 min was used as a positive control (PC). Mean fluorescence value in non-treated MDA-MB-231 cells is set as 1 and relative fluorescence intensity is represented. Data are means ± SD of 4 independent experiments. Statistical significance is assessed by ordinary one-way ANOVA with Tukey's multiple comparisons test. (C) MTT assay was performed on cells treated with 10 mM AA or DHA alone or in the presence of 2000 U/ml catalase, 10 mM NAC or 10 mM GSH for 24 h. Data are means ± SD of at least 6 independent experiments. Statistical significance is assessed by two-way ANOVA with Tukey's multiple comparisons test. ****, P < 0.0001; **, P < 0.01; *, P < 0.05.
Fig. 3
Fig. 3
Extracellular and intracellular H2O2accumulation upon AA treatment in MDA-MB-231 cells. (A) Using Amplex-Red Kit, H2O2 concentration was measured in the medium in the presence of MDA-MB-231 cells, without treatment or treated with 10 mM AA alone, 10 mM AA plus 2000 U/ml catalase, for 5 min, ½ hour and 2 h. The same measurements were done on the medium without cells as controls. Quantifications are means ± SD of 3 independent experiments. (B) Cells transiently expressing respectively cytoplasm-, nucleus-, mitochondria- and cell membrane-targeted HyPer were not treated or treated with 10 mM AA alone or together with 2000 U/ml catalase for 5 min, ½ hour and 2 h. Treatment with 100 μM  H2O2 for 20 min was used as a positive control (PC). HyPer redox state was evaluated by redox Western blot. ox, oxidized form; red, reduced form. Graphs show the quantification of oxidized HyPer (%) vs total HyPer protein. Quantifications are means ± SD of 3 independent experiments. (C) Redox state of PRDX1 and PRDX3 was assessed using redox Western blot in cells without treatment or treated with 10 mM AA alone, 10 mM AA plus 2000 U/ml catalase, for 5 min, ½ hour and 2 h. Treatment with 100 μM  H2O2 for 20 min was used as a positive control (PC). ox, oxidized form; red, reduced form. Quantifications of oxidized PRDX1 or PRDX3 (%) vs total protein in MDA-MB-231 cells are means ± SD of 3 independent experiments. Statistical significance is assessed by ordinary one-way ANOVA with Tukey's multiple comparisons test. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05.
Fig. 4
Fig. 4
Pathway, function and network analysis of the differentially oxidized cysteine-containing proteins identified by “redoxome” upon AA treatment. The alterations of the oxidized proteome of cysteine-containing proteins, after 10 mM AA treatment for ½ hour in MDA-MB-231 cells were analyzed by mass spectrometry. 203 differentially oxidized proteins with P < 0.05 and a fold change over 1.5 in at least one of the two biological experiments were identified between AA-treated and non-treated cells. (A) Significantly altered cysteine-containing proteins were mapped by the IPA analysis software. The top 5 significantly associated canonical pathways, molecular and cellular functions and networks of these proteins are represented. (B) Altered proteins were classified according to their GO biological process pathways in DAVID database. The top significantly (Fisher's modified test) associated biological processes are represented. **, P < 0.01; *, P < 0.05.
Fig. 5
Fig. 5
Effect AA treatment on cell cycles of MDA-MB-231 cells. (A) Non-treated (NT) cells, cells treated for 2 h with 10 mM AA, 10 mM AA plus 2000 U/ml catalase were released in flesh medium. At various time points of post-treatment recovery (from 0 to 24 h), these cells were stained with BrdU for 1 h followed by PI staining and FACS analysis. Dot plots of samples from 0, 6 and 24 h post-treatment recovery are presented with values of the percentages (%) of the population in each phase of cell cycle (G0-G1, S, G2-M). (B) Histogram shows the percentages (%) of cells in G0-G1, S, G2-M phases at 0 h post-AA treatment as means ± SD calculated from 3 independent experiments. Statistical significance is assessed by one-way ANOVA with Tukey's multiple comparisons test. *, P < 0.05. (C) MDA-MB-231 cells were labeled with BrdU for 1 h and then subjected to three treatment conditions for 2 h: non-treatment (NT), treated with 10 mM AA, or 10 mM AA plus 2000 U/ml catalase. Cells were then analyzed by flow cytometry after PI staining at different time points during recovery in growth media (0, 6 or 24 h). Representative graphs of at least 3 experiments are shown.
Fig. 6
Fig. 6
Effect AA treatment on polysome gradient profiles of MDA-MB-231 cells. (A) Non-treated (NT) cells, cells treated for 2 h with 10 mM AA, 10 mM AA plus 2000 U/ml catalase were incubated for 15 min with cycloheximide to stop ribosomal elongation. Cellular RNA was extracted and fractioned in a 15–50% sucrose gradient. Optical density profiles (OD 254 nm) of polysome gradient profiles are shown, and non-polysomal RNA corresponding to fractions 1 to 8 (containing ribosomal RNAs present in 40S, 60S, and 80S ribosomal complexes and free mRNAs) and polysomal RNA corresponding to fractions 9 to 18 (containing mRNAs bound to more than one ribosome) are indicated. (B) The areas under the curve (AUC) of the optical density profiles of non-polysomal and polysomal RNA were measured, the percentage of non-polysomal or polysomal RNA relative to total was then calculated. Quantifications are represented in graphs as means ± SD of 3 independent experiments. Statistical significance is assessed by ordinary one-way ANOVA with Tukey's multiple comparisons test. **, P < 0.01; *, P < 0.05.
Fig. 7
Fig. 7
PRDX1 expression and its link to breast cancer cellular response to AA. (A) PRDX1 mRNA expression patterns in log2 values in 29 breast cancer cells retrieved from publicly available transcriptomic datasets of the Institut Curie breast cancer cell lines. Mean and median values are shown as solid and dashed lines, respectively. (B) Spearman's correlation analysis corresponding to PRDX1 mRNA expression vs IC50 for AA on 13 breast cancer cell lines. IC50 values were calculated from MTT assays for cell viability. Spearman's correlation was performed using GraphPad software. The 7 TNBC cell lines are indicated by green symbols and the 6 non-TNBC cell lines by purple symbols. The best-fit line is in solid orange line and the 95% confidence bands of the best-fit line are indicated in blue lines. Mathematical parameters are represented next to the graphs. (C) logIC50 of AA for breast cancer cell lines and their PRDX1 mRNA expression in log2 values. (D-E-F) MTT assay on HCC-1954, MDA-MB-436 and MDA-MB231 cells transiently transfected with PRDX1 specific siRNA or control siRNA for 48 h followed by treatments with different concentrations of AA for 24 h. The insert is Western blot to verify efficiency of PRDX1 knockdown in siRNA transfected cells. Statistical significance is assessed by two-way ANOVA with Sidak's multiple comparisons test. ****, P < 0.0001.
Fig. 8
Fig. 8
A model for AA- and DHA-induced cytotoxicity in breast cancer cells. Upon AA-treatment, H2O2 is generated in the extracellular medium and then imported to breast cancer cells by aquaporins or by passive diffusion across plasma cellular membranes. Extracellularly generated H2O2 appears to be the main source responsible for pro-oxidant properties of AA at pharmacologic concentrations. Although AA may not be imported to cells from the external environment by sodium-dependent VitC transporters (mainly SVCT2) in breast cancer, AA can be oxidized extracellularly into DHA and then imported through glucose transporters (mainly GLUT1). DHA is reduced to AA intracellularly by GSH-dependent mechanisms, consuming intracellular reduced glutathione. AA thus generated further enhances H2O2 accumulation in different cellular compartments. A substantial increase in H2O2 causes redox modifications of cysteine-containing proteins, including those involved in translation and cell cycle progression. These effects contribute to AA-induced cytotoxicity toward breast cancer cells.
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References

    1. Gorrini C., Harris I.S., Mak T.W. Modulation of oxidative stress as an anticancer strategy. Nat. Rev. Drug Discov. 2013;12(12):931–947. - PubMed
    1. Trachootham D., Alexandre J., Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat. Rev. Drug Discov. 2009;8(7):579–591. - PubMed
    1. Chaiswing L., St Clair W.H., St Clair D.K. Redox paradox: a novel approach to therapeutics-resistant cancer. Antioxid Redox Signal. 2018;29(13):1237–1272. - PMC - PubMed
    1. Du J., Cullen J.J., Buettner G.R. Ascorbic acid: chemistry, biology and the treatment of cancer. Biochim. Biophys. Acta. 2012;1826(2):443–457. - PMC - PubMed
    1. Michels A.J., Frei B. Myths, artifacts, and fatal flaws: identifying limitations and opportunities in vitamin C research. Nutrients. 2013;5(12):5161–5192. - PMC - PubMed

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