Equol exerts anti-tumor effects on choriocarcinoma cells by promoting TRIM21-mediated ubiquitination of ANXA2

Abstract

Choriocarcinoma is a malignant cancer that belongs to gestational trophoblastic neoplasia (GTN). Herein, serum metabolomic analysis was performed on 29 GTN patients and 30 healthy individuals to characterize the metabolic variations during GTN progression. Ultimately 24 differential metabolites (DMs) were identified, of which, Equol was down-regulated in GTN patients, whose VIP score is the 3rd highest among the 24 DMs. As an intestinal metabolite of daidzein, the anticancer potential of Equol has been demonstrated in multiple cancers, but not choriocarcinoma. Hence, human choriocarcinoma cell lines JEG-3 and Bewo were used and JEG-3-derived subcutaneous xenograft models were developed to assess the effect of Equol on choriocarcinoma. The results suggested that Equol treatment effectively suppressed choriocarcinoma cell proliferation, induced cell apoptosis, and reduced tumorigenesis. Label-free quantitative proteomics showed that 136 proteins were significantly affected by Equol and 20 proteins were enriched in Gene Ontology terms linked to protein degradation. Tripartite motif containing 21 (TRIM21), a E3 ubiquitin ligase, was up-regulated by Equol. Equol-induced effects on choriocarcinoma cells could be reversed by TRIM21 inhibition. Annexin A2 (ANXA2) interacted with TRIM21 and its ubiquitination was modulated by TRIM21. We found that TRIM21 was responsible for proteasome-mediated degradation of ANXA2 induced by Equol, and the inhibitory effects of Equol on the malignant behaviors of choriocarcinoma cells were realized by TRIM21-mediated down-regulation of ANXA2. Moreover, β-catenin activation was inhibited by Equol, which also depended on TRIM21-mediated down-regulation of ANXA2. Taken together, Equol may be a novel candidate for the treatment for choriocarcinoma.

Keywords: ANAX2; Choriocarcinoma; Equol; Metabolomics; Proteomics; TRIM21.

Fig. 1

Serum metabolomics reveals different metabolic profiles between normal subjects and patients with GTN. A The OPLS-DA score plot for GTN and normal. B Volcano plot of DMs. C Heatmap of DMs in normal and GTN groups. Log₂FC were calculated to show the differences in levels of metabolites. Those with P < 0.05, VIP ≥ 1, and Log₂FC > 1.5 were considered as the significant DMs between two groups. The threshold of VIP was set to 1. D KEGG enrichment analysis of the biological pathways of DMs. E The 24 identified DMs ranked by their contributions and shown as VIP scores. F A receiver operating characteristics (ROC) curve for Equol level prediction. DMs differential metabolites, GTN gestational trophoblastic neoplasia, VIP variable importance in the projection, OPLS-DA orthogonal projection to latent structures-discriminant analysis

Fig. 2

Effects of Equol on choriocarcinoma cell proliferation and cell cycle. JEG-3 and Bewo cells were treated with different concentrations of Equol for 24 h. A and B CCK-8 assays were performed to detect the growth inhibition of cells. After treatment with Equol (20 or 40 μM) or vehicle for 24 h, C cell cycle distribution was determined by flow cytometry. Veh. vehicle

Fig. 3

Effects of Equol on choriocarcinoma cell apoptosis. JEG-3 and Bewo cells were treated with Equol (20 or 40 μM) or vehicle for 24 h, and then subjected to A Annexin V-FITC/PI apoptosis assays. B The expression of apoptosis-related proteins, including cleaved caspase-3, cleaved caspase-7, cleaved caspase-9, and cleaved PARP, was determined by western blot. C and D The activity of caspase-3 was measured. E JC-1 staining was performed to evaluate the mitochondria membrane potential of cells. F Cytoplasm and mitochondria fraction were subjected to western blot to detect Cyt C. Veh vehicle, Cyt C cytochrome C

Fig. 4

Screening of the proteins regulated by Equol in choriocarcinoma cells. A The PCA plot for the similarity assessment of samples from the Veh. group and the Equol group. B The volcano plot showing the DEPs in the Equol group when compared with the Veh. group. The proteins with P < 0.05 and Log₂FC > 1 were considered as the significant DEPs between two groups. C The sankey diagram combined with a bubble diagram visualized the significantly enriched GO terms associated with protein degradation and the proteins annotated within. D The heatmap visualized the expression patterns of DEPs, and the proteins enriched in the GO terms associated with protein degradation were highlighted. E The binding mode of Equol to TRIM21 by molecule docking. F JEG-3 and Bewo cells were treated with Equol (20 or 40 μM) or vehicle for 24 h. The expression of TRIM21 was measured by western blot. PCA principal component analysis, Veh. vehicle

Fig. 5

Roles of TRIM21 in the effect of Equol on choriocarcinoma cell proliferation and apoptosis. A JEG-3 and Bewo cells were transfected with siTRIM21 or siNC for 48 h. The expression levels of TRIM21 mRNA and protein were measured by qRT-PCR and western blot. At 48 h after cell transfection, the cells were treated with 40 μM Equol for 24 h. B Cell viability was detected by CCK-8 assay. (C) Cell apoptosis was determined by flow cytometry. Veh. vehicle

Fig. 6

Effects of Equol and TRIM21 on the ubiquitination of AXNA2. A The PPI network showing the proteins interacted with TRIM21. B Double immunofluorescence staining of TRIM21 (red) and AXNA2 (green) in JEG-3 and Bewo cells. C Co-immunoprecipitation of TRIM21 with AXNA2 in JEG-3 and Bewo cells. D JEG-3 and Bewo cells were transfected with siTRIM21 or siNC for 48 h, and then treated with 40 μM Equol for 24 h. The expression of AXNA2 was measured by western blot. E JEG-3 and Bewo cells were transfected with siTRIM21 or siNC for 48 h, and 12-h treatment with MG132 (10 μM) was subjected before cell harvesting. F JEG-3 and Bewo cells were treated with 40 μM Equol for 24 h, and 12-h treatment with MG132 (10 μM) was subjected before cell harvesting. E and F The ubiquitination of AXNA2 was detected by co-immunoprecipitation of AXNA2 with ubiquitin. G JEG-3 and Bewo c ells were treated with 40 μM Equol and 10 μM MG132 for 24 h. Western blot analysis of AXNA2 was performed. Veh. Vehicle, Ub ubiquitin

Fig. 7

Roles of ANXA2 in the effect of TRIM21 on the proliferation and apoptosis of Equol-treated choriocarcinoma cells. A JEG-3 and Bewo cells were transfected with siANXA2 or siNC for 48 h. The expression levels of ANXA2 mRNA and protein were measured by qRT-PCR and western blot. JEG-3 and Bewo cells were co-transfected with siTRIM21 and siANXA2 for 48 h, and then treated with 40 μM Equol for 24 h. B Cell viability was detected by CCK-8 assay. C Cell apoptosis was determined by flow cytometry. Veh. vehicle

Fig. 8

Roles of TRIM21 and AXNA2 in Equol-induced effects on β-catenin activation. A JEG-3 and Bewo cells were co-transfected with TOPflash-luc and pRL-TK and treated with 20 or 40 μM Equol for 48 h. B The siTRIM21 or siNC was co-transfected with TOPflash-luc and pRL-TK into JEG-3 and Bewo cells for 48 h, and then the cells treated with 40 μM Equol for 24 h. C JEG-3 and Bewo cells were co-transfected with siTRIM21, siANXA2, TOPflash-luc, and pRL-TK for 48 h, and then treated with 40 μM Equol for 24 h. A–C The relative activity of luciferase was measured. Veh. vehicle. D Schematic illustration of the mechanism of β-catenin activation regulated by Equol

Fig. 9

Equol inhibits tumor growth in vivo. Babl/c nude mice bearing JEG-3-derived tumor xenografts were administrated with Equol (20 mg/kg) or vehicle once a day by gavage. A Tumor volumes, tumor pictures, and tumor weight (TW) results showed that Equol exerted an inhibitory effect on the growth of the JEG-3-derived tumor xenografts. Tumor volume (TV) = 0.5 × a × b²; a: the longest diameter; b: the shortest diameter. B and C Immunohistochemical staining analysis of tumor tissues was performed using anti-TRIM21 and anti-ANXA2 antibodies. Veh. vehicle