Beyond gold: the chemoenhancing mechanism and therapeutic potential of auranofin in melanoma

Abstract

Objective: The objective of the current study was to evaluate the chemosensitizing capacity of auranofin (AF), a gold (I) complex traditionally used in rheumatoid arthritis treatment, in potentiating the cytotoxic effects of doxorubicin (DOX) in melanoma cell models, specifically drug-sensitive (B16F10) and multidrug-resistant (B16F10/ADR) variants.

Methods: Experimental measurements, including in vitro cytotoxicity and apoptosis assays, surface plasmon resonance (SPR), immunoblotting assays, as well as theoretical calculations, such as molecular docking and molecular dynamics (MD) simulations, were used to systematically delineate the interaction dynamics between AF and thioredoxin reductase 1 (TrxR1). The anti-tumor efficacy of co-treatment with AF and DOX was assessed by examining cell viability and apoptotic rates.

Results: Co-treatment with AF and DOX significantly increased anti-tumor efficacy, as evidenced by reduced cell viability and increased apoptotic rates. This synergistic effect was attributed to inhibition of TrxR1 by AF, which compromised tumor cell antioxidant defenses and elevated intracellular reactive oxygen species (ROS), thereby enhancing apoptotic pathways. Notably, AF treatment mitigated the heightened TrxR activity in DOX-resistant cells, intensifying the pro-oxidant effects of DOX, leading to increased ROS production and cell death. The data also showed that AF binds with high affinity to the selenocysteine residue within the catalytic site of TrxR1, which partially overlapped with the binding site of the endogenous substrate, thioredoxin (Trx), but with greater avidity. This unique binding configuration impedes the reduction of Trx by TrxR1, triggering an apoptotic response in cancer cells.

Conclusions: This study underscores the chemosensitizing potential of AF in overcoming multidrug resistance in cancer therapy through redox modulation. The molecular mechanism of action underlying AF on TrxR1 demonstrated the unique binding configuration that impedes the reduction of Trx by TrxR1 and instigates an apoptotic response in cancer cells. These findings pave the way for the clinical application of AF as a chemosensitizer, offering a novel approach to augment the efficacy of existing chemotherapy regimens.

Keywords: Auranofin; TrxR1; anti-cancer; drug resistance; molecular dynamics simulation.

Study Flow

Part I conducts a clinical survival analysis of TrxR1 expression, assessing its correlation with survival in renal, liver, and melanoma cancers using transcriptomic data from HPA via Kaplan-Meier survival curves and the log-rank test, finding that high TrxR1 expression predicts poor survival. Part II focuses on the molecular mechanism of AF-TrxR1 interaction, characterizing AF’s binding to TrxR1 at the selenocysteine active site through molecular docking and MD simulations, revealing a high-affinity Au-Se coordination bond between AF and Sec residue and verified via SPR. Part III performs in vitro validation in melanoma models, including drug-sensitive and acquired DOX-resistant melanoma tumor cells (B16F10/ADR cells), using cytotoxicity (MTT assay), apoptosis, and ROS quantification methods, showing a synergistic effect (CI < 1) and ROS-driven apoptosis. Part IV integrates the mechanistic pathway, elucidating redox dysregulation and apoptotic activation by TrxR1 activity assay and western blot, demonstrating that AF trapped TrxR1 in the oxidized state, blocking Trx reduction and amplifying ROS-induced apoptosis. The study provides a theoretical basis for repurposing auranofin as a chemosensitizer to overcome multidrug resistance in cancer therapy by targeting the TrxR1 redox axis, offering a novel strategy to enhance chemotherapy efficacy. TrxR1, thioredoxin reductase 1; AF, auranofin; DOX, doxorubicin; ROS, reactive oxygen species; MD, molecular dynamics; SPR, surface plasmon resonance; CI, combination index (Chou-Talalay).

Figure 1

Relationship between TrxR1 expression and survival probability of cancer patients. (A) Among renal cancer patients. (B) In liver cancer patients.

Figure 2

Sensitizing effect of AF with DOX for an anti-ADR effect in the B16F10/ADR cell model in vitro. (A) Viability of B16F10 or B16F10/ADR cells exposed to DOX (8 μM), AF (0.5 μM), or DOX plus AF. (B) Intracellular TrxR activity of B16F10 or B16F10/ADR cells exposed to DOX (8 μM), AF (0.5 μM), or DOX plus AF. (C–F) Cytotoxicity of B16F10 or B16F10/ADR cells exposed to DOX (8 μM), AF (0.5 μM), or DOX plus AF. (C) Flow cytometry (FCM) analysis of ROS accumulation. (D) Quantitative analysis of ROS accumulation in panel C. (E) FCM analysis of cell apoptosis. (F) Quantitative analysis apoptosis. Data are presented as the mean ± SD (n = 3). Statistical significance (**P < 0.01; ***P < 0.001) was obtained by two-way ANOVA with LSD multiple comparisons test in A, B, D, and F.

Figure 3

Global docking of AF to TrxR1. (A) Atomic structure of AF, consisting of a triethyl phosphine (2,3,4,6-tetra-O-acetyl-β-1-D-thiopyranosato-S) and sulfur glucose bonded by an Au(I) atom. (B) Conformation of TrxR1 presented by a transparent surface and opaque cartoon. TrxR1 is composed of two homologous chains (chain A in pink and chain B in green). (C) Clustering analysis of the top 100 global docking results. RMSD cut-off for clustering was 4 Å. The cluster with the maximum number of conformers was highlighted in red. There were 19 conformers in this cluster. The position of this cluster was the nearest to the active site of TrxR1. (D) Distribution of the top 100 possible positions of AF on TrxR1. The positions of AF were denoted by the Au atom in AF (orange VDW ball). Red represents the conformers in the biggest cluster.

Figure 4

Local docking of AF to TrxR1. (A) The clustering analysis of top 100 conformers. The RMSD cut-off for clustering was 2 Å. The cluster with the maximum number of conformers is denoted by red. (B) The representative structure in the biggest cluster. The binding free energy of this structure was −4.13 kcal/mol. The Au atom in AF is pink, the Se atom in selenocysteine is yellow, and the distance between Au and Se was 2.59 Å. (C) Sketches of AF bounds with TrxR1. The amino acids and hydrogen bonds in direct contact with AF are depicted using the LIGPLOT program.

Figure 5

MD simulating the interaction between AF and TrxR1. (A) The conformation of AF@TrxR1 complex at 150 ns. The right panel is the zoom-in of the red square region of the left panel. Chain A is pink and chain B is green. The Ala26, Leu112, and Ile347 residues had hydrophobic interactions with AF are shown in black. Lys29 and Gln348 formed hydrogen bonds with AF are shown in blue and red, respectively. (B) The time-dependent distance between the Au atom of AF and the Se atom of Sec498 in TrxR1. (C) The RMSD of TrxR1 (black line) and RMSD of the residues close to AF (red line) after interacting with AF. (D) The number of contact atoms, total contact (black), and hydrophobic contact (red). (E) The total number of hydrogen bonds formed in the AF@TrxR1 complex (black square); the number of hydrogen bonds formed in AF/Lys29 or AF/Gln348 (red dot).

Figure 6

Global docking of AF to mutated TrxR1. (A) Clustering analysis of the top 100 conformers. The RMSD cut-off for clustering was 4 Å. The cluster with the maximum number of conformers is highlighted in red. There were 7 conformers in this cluster. (B) Distribution of the top 100 possible positions of AF on mutated TrxR1. The positions of AF are denoted by the Au atom in AF (yellow VDW ball). Red represents the active site of TrxR1 targeted.

Figure 7

Inhibition of AF to the formation of TrxR1/Trx complex. (A) Conformation of the TrxR1/Trx (left) and AF@TrxR1/Trx complexes (right). The pink and green cartoon modes were the two chains of TrxR1 and the cyan cartoon chain was Trx. AF was shown as a VDW ball. (B) Residues in direct contact with each other in the Trx/TrxR1 (left) and AF@TrxR1/Trx systems (right) were highlighted and visualized by the LIGPLOT+ program. (C) The COM distance between TrxR1 and Trx in the TrxR1/Trx (black line) and AF@TrxR1/Trx systems (red line). (D) The interaction energy between the TrxR1 and Trx in TrxR1/Trx (black line) and AF@TrxR1/Trx systems (red line).

Figure 8

The affinities of AF/TrxR1 and Trx/TrxR1 systems based on the SPR assay. (A) The SPR assay of the AF/TrxR1 system. A series of AF concentrations (7.81 nM–250 nM) were passed over the TrxR1-immobilized sensor chip to obtain the affinity measurement. (B) The SPR assay of the Trx/TrxR1 system. A series of Trx concentrations (0.44 μM–14.25 μM) were passed over the TrxR1-immobilized sensor chip. The SPR experiments were independently repeated three times. (C) Western blot detection of the specific protein expression involved in reducing ADR. Tubulin was used as a loading control. (D) Quantitative analysis of protein expression. The relative protein expression was normalized to the control group. Data are presented as the mean ± SD (n = 3). The statistical significance (**P < 0.01) was obtained by two-way ANOVA with LSD multiple comparisons.

Figure 9

Mechanism underlying AF enhances the cytotoxic effects of DOX in melanoma cells. Auranofin (AF) exerts its effect by targeting the redox-active site of thioredoxin reductase 1 (TrxR1). As illustrated in the left panel, AF is represented as a molecular structure interacting with the catalytic site of TrxR1. TrxR1, shown as a new cartoon with its active site containing a selenocysteine (Sec) residue. The two-way arrow connects the selenium (Se) atom of the Sec residue and the Au atom in the AF molecule. This catalytic site is constituted by Lys29 (blue), Gln348 (red), and Leu112 (black). It has been demonstrated that AF binds with high affinity to the Sec residue within the catalytic site of TrxR1, partially overlapping with the binding site of the endogenous substrate, thioredoxin (Trx), but with greater avidity. This binding impedes the reduction of Trx by TrxR1. The right panel is the mechanistic pathway. Normally, NADPH oxidation to NADP⁺ supplies electrons via FAD to the TrxR1 redox center, reducing the Cys-Sec bond to the -SH/-SeH groups. Reduced TrxR1 then restores Trx to its active form, scavenging doxorubicin-induced reactive oxygen species (ROS). AF blocks this cascade by trapping TrxR1 in its oxidized state, preventing Trx reduction and causing ROS accumulation that triggers apoptosis, synergistically enhancing chemotherapeutic efficacy.