Auranofin inhibits glucose uptake and induces ferroptosis in anaplastic thyroid cancer

doi:10.11958/20251716

Abstract

Abstract:

Objective To investigate the effect and molecular mechanism of auranofin (AUF) in inhibiting glucose uptake and inducing ferroptosis in anaplastic thyroid carcinoma (ATC) cells. Methods CAL-62 cells were routinely cultured and treated with AUF at various concentrations (0, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 μmol/L). Cells were also treated with 0.6 μmol/L AUF (AUF group), BAY (BAY group), the combination of 0.6 μmol/L AUF and BAY (AUF+BAY group), Erastin (Erastin group) and the combination of 0.6 μmol/L AUF and Erastin (AUF+Erastin group). Untreated cells were served as the control group. Cell viability of CAL-62 cells was detected by CCK-8 assay, and cell apoptosis was determined by flow cytometry. Glucose uptake was measured using a glucose detection kit, and intracellular iron content and glutathione (GSH) levels were determined using ferroptosis-related assay kits. The mRNA expressions of glucose transporter 1 (GLUT1), long-chain acyl-CoA synthetase 4 (ACSL4) and glutathione peroxidase 4 (GPX4) in ATC tissue were detected by quantitative real-time polymerase chain reaction (qRT-PCR). A subcutaneous xenograft tumor model was established in nude mice to evaluate the antitumor effect of AUF on ATC in vivo. The protein expression levels of ACSL4, GPX4 and thioredoxin reductase 1 (TXNRD1) were detected by Western blot assay. Results Compared with the control group, AUF inhibited the viability of CAL-62 cells in a dose-dependent manner (P<0.01). Both AUF alone and its combined treatment with BAY or Erastin increased the apoptosis rate (P<0.05). Compared with the control group, the residual glucose content in the cell culture supernatant and intracellular iron content were increased, while the GSH content was decreased in the 0.8 and 1.0 μmol/L AUF groups (P<0.05). Compared with the control group, the mRNA expression levels of GLUT1 and GPX4 were decreased in the 0.6, 0.8 and 1.0 μmol/L AUF groups (P<0.05), and the mRNA expression level of ACSL4 was increased in the 0.8 and 1.0 μmol/L AUF groups (P<0.05). In animal experiments, the tumor volume was significantly smaller in the AUF+Erastin group than that in the control group only on day 14 (P<0.05), while there was no statistically significant difference in tumor volume between the AUF group and the control group (P>0.05). There was no statistically significant difference in body weight between each group and the control group (P>0.05). Compared with the control group, the protein expression levels of TXNRD1 and GPX4 were decreased and the protein expression level of ACSL4 was increased in the AUF group (P<0.05). The protein expression level of GPX4 was decreased and the protein expression level of ACSL4 was increased in the AUF+Erastin group (P<0.05). Conclusion AUF promotes ferroptosis and suppresses glucose uptake in ATC cells, thereby inhibiting their proliferation.

Key words: auranofin, thyroid carcinoma, anaplastic, ferroptosis, glucose uptake, TXNRD1, GLUT1

CLC Number:

R736

Figures/Tables 12

References 22

[1]	RAO S N, SMALLRIDGE R C. Anaplastic thyroid cancer:An update[J]. Best Pract Res Clin Endocrinol Metab, 2023, 37(1):101678. doi:10.1016/j.beem.2022.101678.
[2]	ALOBUIA W, GILLIS A, KEBEBEW E. Contemporary management of anaplastic thyroid cancer[J]. Curr Treat Options Oncol, 2020, 21(10):78. doi:10.1007/s11864-020-00776-2.
[3]	ZHOU Q, MENG Y, LI D, et al. Ferroptosis in cancer:from molecular mechanisms to therapeutic strategies[J]. Signal Transduct Target Ther, 2024, 9(1):55. doi:10.1038/s41392-024-01769-5.
[4]	LI D, LI Y. The interaction between ferroptosis and lipid metabolism in cancer[J]. Signal Transduct Target Ther, 2020, 5(1):108. doi:10.1038/s41392-020-00216-5.
[5]	CHEN H, PENG F, XU J, et al. Increased expression of GPX4 promotes the tumorigenesis of thyroid cancer by inhibiting ferroptosis and predicts poor clinical outcomes[J]. Aging, 2023, 15(1):230-245. doi:10.18632/aging.204473.
[6]	CHEN J, DING C, CHEN Y, et al. ACSL4 reprograms fatty acid metabolism in hepatocellular carcinoma via c-Myc/SREBP1 pathway[J]. Cancer Lett, 2021, 502:154-165. doi:10.1016/j.canlet.2020.12.019.
[7]	CHANDEL N S. Glycolysis[J]. Cold Spring Harb Perspect Biol, 2021, 13(5):a040535. doi:10.1101/cshperspect.a040535.
[8]	ANCEY P B, CONTAT C, BOIVIN G, et al. GLUT1 expression in tumor-associated neutrophils promotes lung cancer growth and resistance to radiotherapy[J]. Cancer Res, 2021, 81(9):2345-2357. doi:10.1158/0008-5472.Can-20-2870.
[9]	YAMASHITA M. Auranofin:past to present, and repurposing[J]. Int Immunopharmacol, 2021, 101(Pt B):108272. doi:10.1016/j.intimp.2021.108272.
[10]	YAN X, ZHANG X, WANG L, et al. Inhibition of thioredoxin/thioredoxin reductase induces synthetic lethality in lung cancers with compromised glutathione homeostasis[J]. Cancer Res, 2019, 79(1):125-132. doi:10.1158/0008-5472.Can-18-1938.
[11]	TIEDJE V, STUSCHKE M, WEBER F, et al. Anaplastic thyroid carcinoma:review of treatment protocols[J]. Endocr Relat Cancer, 2018, 25(3):R153-R161. doi:10.1530/erc-17-0435.
[12]	SAINI S, TULLA K, MAKER A V, et al. Therapeutic advances in anaplastic thyroid cancer:a current perspective[J]. Mol Cancer, 2018, 17(1):154. doi:10.1186/s12943-018-0903-0.
[13]	MADEIRA J M, GIBSON D L, KEAN W F, et al. The biological activity of auranofin:implications for novel treatment of diseases[J]. Inflammopharmacology, 2012, 20(6):297-306. doi:10.1007/s10787-012-0149-1.
[14]	GAMBERI T, CHIAPPETTA J, FIASCHI T, et al. Upgrade of an old drug:Auranofin in innovative cancer therapies to overcome drug resistance and to increase drug effectiveness[J]. Med Res Rev, 2022, 42(3):1111-1146. doi:10.1002/med.21872.
[15]	LIU C, ZHAO Y, WANG J, et al. FoxO3 reverses 5-fluorouracil resistance in human colorectal cancer cells by inhibiting the Nrf2/TR1 signaling pathway[J]. Cancer Lett, 2020, 470:29-42. doi:10.1016/j.canlet.2019.11.042.
[16]	ZHANG Z, LI X, YANG F, et al. DHHC9-mediated GLUT1 S-palmitoylation promotes glioblastoma glycolysis and tumorigenesis[J]. Nat Commun, 2021, 12(1):5872. doi:10.1038/s41467-021-26180-4.
[17]	CAI K, CHEN S, ZHU C, et al. FOXD1 facilitates pancreatic cancer cell proliferation,invasion,and metastasis by regulating GLUT1-mediated aerobic glycolysis[J]. Cell Death Dis, 2022, 13(9):765. doi:10.1038/s41419-022-05213-w.
[18]	WU Q, BA-ALAWI W, DEBLOIS G, et al. GLUT1 inhibition blocks growth of RB1-positive triple negative breast cancer[J]. Nat Commun, 2020, 11(1):4205. doi:10.1038/s41467-020-18020-8.
[19]	李艳萍, 王协涛, 史立彬, 等. 白藜芦醇调控Nrf2-GPX4通路对H₂O₂诱导肺泡上皮细胞铁死亡的影响[J]. 天津医药, 2023, 51(6):568-572.
	LI Y P, WANG X T, SHI L B, et al. Influence of resveratrol on H₂O₂-induced ferroptosis in alveolar epithelial cells by regulating the Nrf2-GPX4 pathway[J]. Tianjin Med J, 2023, 51(6):568-572. doi:10.11958/20221584.
[20]	ZHANG W, LIU Y, LIAO Y, et al. GPX4,ferroptosis,and diseases[J]. Biomed Pharmacother, 2024, 174:116512. doi:10.1016/j.biopha.2024.116512.
[21]	CHEN X, LI J, KANG R, et al. Ferroptosis:machinery and regulation[J]. Autophagy, 2020, 17(9):2054-2081. doi:10.1080/15548627.2020.1810918.
[22]	CHEN C, YANG Y, GUO Y, et al. CYP1B1 inhibits ferroptosis and induces anti-PD-1 resistance by degrading ACSL4 in colorectal cancer[J]. Cell Death Dis, 2023, 14(4):271. doi:10.1038/s41419-023-05803-2.

基因名称	引物序列（5'→3'）	产物大小/bp
GLUT1	上游：GGCCAAGAGTGTGCTAAAGAA 下游：ACAGCGTTGATGCCAGACAG	201
ACSL4	上游：CATCCCTGGAGCAGATACTCT 下游：TCACTTAGGATTTCCCTGGTCC	96
GPX4	上游：GAGGCAAGACCGAAGTAAACTAC 下游：CCGAACTGGTTACACGGGAA	100
GAPDH	上游：GGAGCGAGATCCCTCCAAAAT 下游：GGCTGTTGTCATACTTCTCATGG	197

基因名称	引物序列（5'→3'）	产物大小/bp
GLUT1	上游：GGCCAAGAGTGTGCTAAAGAA 下游：ACAGCGTTGATGCCAGACAG	201
ACSL4	上游：CATCCCTGGAGCAGATACTCT 下游：TCACTTAGGATTTCCCTGGTCC	96
GPX4	上游：GAGGCAAGACCGAAGTAAACTAC 下游：CCGAACTGGTTACACGGGAA	100
GAPDH	上游：GGAGCGAGATCCCTCCAAAAT 下游：GGCTGTTGTCATACTTCTCATGG	197

组别	葡萄糖浓度/（n=4，mmol/L）	铁含量/（n=3，ng/10⁴ cell）	GSH含量/（n=4，μg/10⁶prot）
对照组	8.60±1.64	3.92±0.49	1.32±0.02
0.6 μmol/L AUF组	13.23±2.58	6.86±1.77	0.89±0.01^a
0.8 μmol/L AUF组	23.44±3.71^ab	8.16±1.41^a	0.99±0.21^a
1.0 μmol/L AUF组	34.83±3.28^abc	11.59±2.26^ab	0.68±0.01^ac
F	64.160^＊＊	11.590^＊＊	24.810^＊＊

组别	葡萄糖浓度/（n=4，mmol/L）	铁含量/（n=3，ng/10⁴ cell）	GSH含量/（n=4，μg/10⁶prot）
对照组	8.60±1.64	3.92±0.49	1.32±0.02
0.6 μmol/L AUF组	13.23±2.58	6.86±1.77	0.89±0.01^a
0.8 μmol/L AUF组	23.44±3.71^ab	8.16±1.41^a	0.99±0.21^a
1.0 μmol/L AUF组	34.83±3.28^abc	11.59±2.26^ab	0.68±0.01^ac
F	64.160^＊＊	11.590^＊＊	24.810^＊＊

组别	GLUT1	GPX4	ACSL4
对照组	1.00±0.00	1.00±0.00	1.00±0.00
0.6 μmol/L AUF组	0.59±0.19^a	0.75±0.08^a	1.14±0.07
0.8 μmol/L AUF组	0.44±0.05^a	0.40±0.06^ab	1.48±0.05^ab
1.0 μmol/L AUF组	0.62±0.10^a	0.79±0.05^ac	1.71±0.17^ab
F	13.590^＊＊	56.640^＊＊	33.950^＊＊