Auranofin as a Novel Anticancer Drug for Anaplastic Thyroid Cancer

Abstract

Background/Objectives: Anaplastic thyroid cancer (ATC) is an aggressive and rare cancer with a poor prognosis, and traditional therapies have limited efficacy. This study investigates drug repositioning, focusing on auranofin, a gold-based drug originally used for rheumatoid arthritis, as a potential treatment for ATC. Methods: Auranofin was identified from an FDA-approved drug library and tested on two thyroid cancer cell lines, 8505C and FRO. Antitumor efficacy was evaluated through gene and protein expression analysis using Western blot, FACS, and mRNA sequencing. In vivo experiments were conducted using subcutaneous injections in nude mice to confirm the anticancer effects of auranofin. Results: Auranofin induced reactive oxygen species (ROS) production and apoptosis, leading to a dose-dependent reduction in cell viability, G1/S phase cell cycle arrest, and altered expression of regulatory proteins. It also inhibited cancer stem cell activity and suppressed epithelial-mesenchymal transition. mRNA sequencing revealed significant changes in the extracellular matrix-receptor interaction pathway, supported by Western blot results. In vivo xenograft models demonstrated strong antitumor activity. Conclusions: Auranofin shows promise as a repurposed therapeutic agent for ATC, effectively inhibiting cell proliferation, reducing metastasis, and promoting apoptosis. These findings suggest that auranofin could play a key role in future ATC treatment strategies.

Keywords: anaplastic thyroid cancer; auranofin; chemotherapy; drug repositioning.

Figure 1

Screening of an FDA-approved library and auranofin as a potent drug for thyroid cancer treatment. (A) Schematic image of the screening strategy. (B) The cell viability was assessed by CCK-8 assay. Four drugs that showed <50% reduction in cell viability (% of control) at a concentration of 10 μM in 8505C and FRO cells. (C) Chemical structures of auranofin, idarubicin, mitoxantrone, and nisoldipine. (D) IC50 values of auranofin, mitoxantrone, and idarubicin were determined from a panel of 772 drugs. Cell viability decreased in a dose-dependent manner with increasing concentrations of each drug. (E) 8505C, FRO, and human thyroid fibroblasts were treated with auranofin (0, 0.001, 0.01, 0.1, 1, 5, 10 μM) or control for 24 h. The results are presented as means ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

(A) Colony formation assay using 8505C and FRO cells was performed as described in the Materials and Methods Section (Section 3) (magnification, ×400). (B) Auranofin induces cell cycle arrest. Cells were treated with auranofin for 24 h, then analyzed by flow cytometry after PI staining. (C) The expression of G1/S phase cell cycle-associated proteins after treatment with auranofin. Whole lysates were subjected to Western blot analysis. The results are presented as means ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Auranofin induces ROS production and apoptosis in thyroid cancer cells. (A) Reactive oxygen species (ROS) were measured using flow cytometry with DCF-DA assay in thyroid cancer cells. Cells were treated with 3 μM auranofin with or without 1 mM NAC for 24 h and then reacted with DCF-DA for 24 h. (B) Auranofin induces apoptosis. Representative images of cell apoptosis stained with Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI). Cells were treated with auranofin with or without 1 mM NAC for 24 h, then stained with Annexin V-FITC/PI, and analyzed by cytometry as described in the Materials and Methods Section (Section 3). (C) Expression of markers of apoptosis evaluated by Western blot analysis. Cells were treated with auranofin for 24 h. Western blot analysis showing the expression levels of cleaved PARP, BAX, and Bcl-2 in treated cells. The results are presented as means ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Functional enrichment analysis of common downregulated genes using GOBP and KEGG analysis. (A). Volcano plot of DEGs between control and auranofin-treated cells. (B). GOBP (gene ontology biological process) enrichment analysis on the commonly down regulated genes. The x-axis is the percentage of genes in the graph belonging to a pathway (fold enrichment). The y-axis is GOBP term. KEGG pathway enrichment analysis on the commonly downregulated genes. The volcano plot of the DEGs is shown in Figure 4A. (C). Validation of 5 downregulated differentially expressed genes using RT-PCR. The results are presented as means ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Auranofin suppresses invasion and sphere formation of thyroid cancer cells. (A) Effects of auranofin on thyroid cancer cell migration and invasion. Transwell migration and invasion assays were performed to investigate the effects of auranofin on the migration and invasion abilities of 8505C and FRO cell lines. The relative number of invaded or migrated cells following treatment with 1 and 3 μM of auranofin. Both migration and invasion were significantly decreased when compared to those of control cells. (B) Auranofin suppresses the expression of epithelial-to-mesenchymal transition marker proteins. The 8505C and FRO cells were treated with DMSO (Control) and 1 and 3 μM of auranofin. Whole-cell protein extracts were analyzed after 24 h of treatment by Western blot with the indicated antibodies. (C) Auranofin suppresses sphere formation. The 8505C and FRO cells were treated with DMSO (Control) and 1 and 3 μM of auranofin. All cells were grown in ultra-low attachment plates for 2 weeks. Quantification of the number and diameter of spheres (n = 3). The results are presented as means ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Treatment with auranofin inhibits tumor growth in a xenograft mouse model. (A) Anti-tumor efficacy of auranofin in the FRO xenograft mouse model. BALB/c mice (n = 5) were treated with PBS (Control) at 100 μM. Tumor volumes were recorded every 3 days. (B) Photographs of tumors in each group. Mice were sacrificed after 25 days of auranofin treatment. (C) Comparison of the final tumor weight and mouse weight in each group. (D) The tumor sections were subjected to IHC staining using antibodies against BAX and PCNA. (E,F) RT-PCR and Western blot analysis of changes in E-cad, Slug, ITGB8, ITGB1, PCNA, and BAX expression in tumor xenografts of mice treated with auranofin. The results are presented as means ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.