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. 2022 Jun:52:102294.
doi: 10.1016/j.redox.2022.102294. Epub 2022 Mar 22.

Redox proteome analysis of auranofin exposed ovarian cancer cells (A2780)

Giovanni Chiappetta  1 , Tania Gamberi  2 , Fiorella Faienza  3 , Xhesika Limaj  4 , Salvatore Rizza  5 , Luigi Messori  6 , Giuseppe Filomeni  7 , Alessandra Modesti  8 , Joelle Vinh  4
Affiliations

Redox proteome analysis of auranofin exposed ovarian cancer cells (A2780)

Giovanni Chiappetta et al. Redox Biol. 2022 Jun.

Abstract

The effects of Auranofin (AF) on protein expression and protein oxidation in A2780 cancer cells were investigated through a strategy based on simultaneous expression proteomics and redox proteomics determinations. Bioinformatics analysis of the proteomics data supports the view that the most critical cellular changes elicited by AF treatment consist of thioredoxin reductase inhibition, alteration of the cell redox state, impairment of the mitochondrial functions, metabolic changes associated with conversion to a glycolytic phenotype, induction of ER stress. The occurrence of the above cellular changes was extensively validated by performing direct biochemical assays. Our data are consistent with the concept that AF produces its effects through a multitarget mechanism that mainly affects the redox metabolism and the mitochondrial functions and results into severe ER stress. Results are discussed in the context of the current mechanistic knowledge existing on AF.

Keywords: Auranofin; Cysteine; Gold drugs; Ovarian cancer; Redox proteomics.

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Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Fig. 1 (A) Cell viability time course upon auranofin treatment using MTT assay. Values were obtained by measuring the percentage of treated-A2780 viable cells relative to untreated controls after 6, 12, 24, 48, and 72 h of incubation with AF 72 h-exposure IC50-dose. (B) TrxR enzyme inhibition time course assay was performed at 6, 12 and 24 h of treatment using a commercial thioredoxin reductase assay kit (Sigma-Aldrich). All the results are reported as mean ± SD of at least three independent biological experiments analyzed in triplicate. The statistical analysis was carried out by a two-tailed T-test using Graphpad Prism v 6.0 (*p < 0.05, ****p < 0.0001).
Fig. 2
Fig. 2
Workflow of the cysteine redox proteomics workflow. In the step A, reduced thiols (-SH) are alkylated with a high concentration of iodoacetamide as showed in previous studies [[41], [42]]. In the step B, after removing IAM, reversible thiol oxidations are reduced by DTT. In the step C, after DTT removal, the newly generated reduced thiols form a mixed disulfide with biotin-HPDP. In step D, the proteins are trypsin digested and loaded onto a streptavidin resin. In step E, biotin-HPDP labeled peptides are eluted by DTT cleaving the biotin arm. The bound and unbound peptide fractions (panel F) are analyzed by LC-MSMS (panel G) in separate runs.
Fig. 3
Fig. 3
Volcano plot of the protein expression profiles. Positive values correspond to protein upregulation. Red dots in the white region of the graphic are considered statistically significant fold changes. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4
Fig. 4
Enriched protein networks obtained with Ingenuity Pathway Analysis. Performing the network analysis in the Ingenuity framework, Ribonucleoside Reductase (RNR) resulted in being at the center of the network of a cluster of upregulated proteins (panel A). RNR1 and RNR2 resulted upregulated in the Auranofin treated samples as shown in panel A, where the histograms represent the fold change (Log2) of RNRs expression compared to the untreated samples., Caspase 3 resulted to be at the center of the second top-ranked enriched network pointing to the caspase-mediated apoptosis (panel B).
Fig. 5
Fig. 5
Volcano plots of the reduced and oxidized Cys fold changes. Fold changes were normalized by protein expression levels. Quantitative proteomics data were obtained from three biological replicates. Panel C: Chemical-physical features of the potential oxidized cysteines from Cpipe analysis. Cpipe calculates a probability score (values between 0 and 1) evaluating the tendency of a Cys residue to exhibit a given chemical-physical feature. We built a background list (control) containing the Cpipe scores for all Cys residues detected in our proteomic analysis (when a PDB file was available). Then, we calculated for the background list and for the oxidized cysteine list the percentage of Cys residues exhibiting a score >0.75 for the tendency to be post-translational modified (PTM), to be exposed (exposition), to bind metals (metal binding), to be nucleophile (energy) and to form disulfide bonds (disulfide). A higher percentage of cysteine residues found with increased oxidized level (in orange) after AF treatment showed a PTM score >0.75 compared to the background list. A lower percentage showed metal-binding scores >0.75. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 6
Fig. 6
(A) Fold changes measured for some Trx and GSH pathway proteins. (B) Fold changes normalized by protein expression for some oxidized/reduced cysteines belonging to TRX and GSH pathway proteins.
Fig. 7
Fig. 7
(A) Western blot analysis of some OXPHOS subunits. Representative Immunoblots are shown together with the corresponding Coomassie-stained PVDF membranes. Histogram reports normalized mean relative-integrated-density ± SD values of the OXPHOS bands. The statistical analysis was carried out using one-way ANOVA test followed by Tuckey's multiple comparisons test using Graphpad Prismv v.6.0 (*p < 0.05). (B) Oxygen consumption rate measured by Clark's electrode and lactate production. Histograms report the mean values ± SD of at least three independent experiments. The statistical analysis was carried out by a two-tailed T-test using Graphpad Prism v 6.0 (*p < 0.05, ****p < 0.0001). (C) Cell death analysis of A2780 cells transfected with WT or C501S HA-TRAP1. Dead cells were measured by flow cytometry evaluating the percentage of cells in Sub-G1 population following Propidium Iodide staining. The histogram shows fold change (AF treated versus untreated cells) in the number of dead cells as mean ± s.e.m. of three independent experiments. Significance is **, p < 0,01 (p=0,0056) by two-tailed Student's T test. (D) PARP1 cleavage was evaluated in WT and C501S A2780 cells by Western blot analysis with anti-PARP1, anti-TRAP1 and anti-LDH antibodies (E) Schematic representation of IMS folding machinery. Blue icons represent downregulated proteins in AF-treated cells. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 8
Fig. 8
Kegg's pathway representation of the Protein Processing in ER pathway. The list of upregulated protein was submitted to the bionformatics tool DAVID. Protein Processing in ER was one of the enriched Kegg's pathways (Benjamini corrected p-value = 0.01). The proteins highlighted by a red star were found upregulated. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 9
Fig. 9
(A). Ubiquitinated protein level. Representative Immunoblots are shown together with the corresponding Coomassie-stained PVDF membranes. Histogram reports normalized mean relative-integrated-density ± SD values of the ubiquitinated protein bands. The statistical analysis was carried out by a two-tailed T-test using Graphpad Prism v 6.0. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (B) Bax and Bcl-2 protein amount. Representative Immunoblots are shown together with the corresponding Coomassie-stained PVDF membranes. (C) Immunoblots of GRP78, ERp44 and ERO1 in control and AF treated A2780 cells. (D) Schematic representation of ER/mitochondria cross-talk highlighted by our proteomics results. Red icons represent up-regulated proteins.
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