F-Spondin Is the Signal by Which 2-Methoxyestradiol Induces Apoptosis in the Endometrial Cancer Cell Line Ishikawa

Abstract

The metabolite 2-methoxyestradiol (2ME) is an endogenous estrogen metabolite with potential therapeutic properties in reproductive cancers. However, the molecular mechanisms by which 2ME exerts its anticancer activity are not well elucidated. The purpose of this study was to determine the molecular signals associated with the apoptotic effects of 2ME in a human endometrial cancer cell line. Ishikawa cells were treated with non-apoptotic (0.1 µM) or apoptotic concentrations (5 µM) of 2ME, and 12 hours later mRNA levels for Scd2, Snx6, and Spon1 were determined by real-time PCR. We then investigated by immunofluorescence and Western blot the expression and distribution of F-spondin, encoded by Spon1, in Ishikawa cells treated with 2ME 5 µM at 6, 12, or 24 h after treatment. The role of estrogen receptors (ER) in the effect of 2ME on the Spon1 level was also investigated. Finally, we examined whether 2ME 5 µM induces cell death in Ishikawa cells pre-incubated with a neutralizing F-spondin antibody. Non-apoptotic or apoptotic concentrations of 2ME decreased Scd2 and increased Snx6. However, Spon1 was only increased with the 2ME apoptotic concentration. F-spondin protein was also increased at 12 and 24 h after 2ME treatment, while 2ME-induced Spon1 increase was independent of ER. Neutralization of F-spondin blocked the effect of 2ME on the cell viability. These results show that F-spondin signaling is one of the components in the apoptotic effects of 2ME on Ishikawa cells and provide experimental evidence underlying the mechanism of action of this estrogen metabolite on cancer cells.

Sorting nexins: A novel promising therapy target for cancerous/neoplastic diseases

“…However, very few studies have addressed the possible mechanisms of SNX6 in modulating cancerous/neoplastic diseases. Rincón‐Rodriguez et al (2019) have demonstrated that SNX6 is a target gene of the metabolite 2‐methoxyestradiol (2ME) that is an endogenous estrogen metabolite with potential therapeutic properties in reproductive cancers. They have further found that SNX6 is responding to 2ME both in apoptotic and nonapoptotic concentrations, suggesting the suppressor role of SNX6 in endometrial cancer (Rincon‐Rodriguez et al, 2019).…”

Identification of a Novel Oncogenic Fusion Gene SPON1-TRIM29 in Clinical Ovarian Cancer That Promotes Cell and Tumor Growth and Enhances Chemoresistance in A2780 Cells

“…While SPON1 expression is found in malignancies including ovarian cancer [ 44 ], the effect of SPON1 depends on the cancer type. In osteosarcoma cells, SPON1 promotes cell migration and invasion [ 45 ], whereas SPON1 exhibits an antioncogenic roles in some tumors: SPON1 expression is negatively correlated with survival in bladder cancer [ 46 ], SPON1 mediates the apoptotic effect of anticancer reagent in uterine endometrial cancer cells [ 47 ], and SPON1 suppression leads to proliferation, migration, and invasion in hepatocellular carcinoma [ 48 ]. In the putative fusion protein based on the SPON1-TRIM29 transcript, it contains 115 amino acids of SPON1 N-terminal signal sequence, whereas it lacks the rest of a large region containing reeler, spondin N, and thrombospondin type1 domains, which are conserved in spondin-like extracellular matrix proteins [ 49 ].…”

Hydroethanolic Extracts of the Aristotelia Chilensis (Maqui) Berry Reduces Cellular Viability and Invasiveness in the Endometrial Cancer Cell Line Ishikawa

“…in PI channel. 2-methoxiestradiol (2ME) 5 µM was used as positive control 26 , 27 and untreated cells were used as control. The percentages for each cell cycle stage were analyzed by the ModFit LT program (v5.0.9, Verity Software House), and the subG1 population was excluded from the histogram because the purpose of this assay was the analysis of cell cycle.…”

“…to analyze viable (double negative), early apoptotic (Annexin V positive), late apoptotic (Annexin V positive, PI positive), and necrotic (PI positive) populations in BD Accuri C6 software (version 1.0.264.21, Accuri ® cytometers, Inc.). As positive control we used 2ME 5 µM 26 , 27 and untreated cells were used as control.…”

“…in PI channel. 2-methoxiestradiol (2ME) 5 µM was used as positive control 26,27 and untreated cells were used as control.…”