Salinomycin modulates viability, invasion, and inflammatory responses in primary endometriotic stromal cell cultures in association with Wnt/β-catenin signaling

Endometriosis is a chronic inflammatory disorder characterized by aberrant stromal-cell behavior, including enhanced invasion, resistance to apoptosis, and sustained cytokine production. Dysregulation of Wnt/β-catenin signaling has been linked to these pathological features, emphasizing its association with disease-related cellu - lar phenotypes. Identifying non-hormonal approaches that selectively target endometriotic stromal cells remains an important research focus. In this study, primary endometriotic stromal cell cultures derived from ectopic lesions of women with ovarian endometrioma were compared with primary control endometrial stromal cell cultures from women without endometriosis. Vimentin and CK18 were evaluated in separate flow-cytometry tubes; therefore, the designation of these cultures as stromal cultures is operational and does not imply com - plete lineage purity. Cells were treated with Salinomycin, and MTT-derived viable-cell/metabolic activity, Matrigel invasion, cytokine secretion, and selected molecular markers related to Wnt/β-catenin signaling, cell- cycle regulation, invasion, and apoptosis were evaluated using MTT assays, Transwell assays, ELISA, and quantitative real-time PCR. Salinomycin induced a dose- and time-dependent reduction in MTT signal, with primary endometriotic cultures showing greater sensitivity than control cultures. At the selected experimental concentration, salinomycin reduced the viable-cell/metabolic signal and invasion in primary endometriotic stromal cell cultures and decreased IL-6 and IL-8 secretion. These phenotypic changes were accompanied by increased β-catenin phosphorylation at Ser45, reduced AXIN2, Cyclin D1, and MMP-9 expression, and increased Caspase-3 expression in ectopic cultures. Because MTT alone cannot distinguish reduced prolifera - tion from cytotoxicity or altered metabolism, the findings are interpreted as viability-associated and prolif - eration-associated responses rather than direct proof of isolated growth arrest. These findings indicate that Salinomycin modulates disease-relevant features of primary endometriotic stromal cell cultures in association with Wnt/β-catenin pathway-related changes and support further mechanistic investigation of this experimental non-hormonal approach. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-67394-0

Endometriosis · Salinomycin · Wnt/β-catenin signaling · Endometriotic stromal cells · Inflammation · Cell invasion

Endometriosis is a chronic, estrogen-dependent inflammatory disorder in which endometrial-like tissue devel - ops outside the uterine cavity 1,2. Although the disease is histologically benign, endometriotic lesions can show several tumor-like biological features, including adhesion, invasion, remodeling of surrounding tissue, and per - sistence at ectopic sites3. These biological properties are clinically important because endometriosis is frequently associated with chronic pelvic pain, dysmenorrhea, dyspareunia, abnormal uterine bleeding, and infertility, all of which may substantially impair quality of life 4. The origin and progression of the disease are not explained by a single mechanism. Retrograde menstruation remains a major theory, but lesion establishment also depends on immune dysfunction, altered proliferation and apoptosis, aberrant endocrine responses, and genetic susceptibil - ity2,5. In advanced disease, pelvic adhesions and fibrosis are common and may reflect repeated cycles of tissue injury and repair within ectopic lesions6,7. Among the signaling pathways implicated in endometriosis, Wnt/β-catenin signaling has received increas - ing attention. Wnt signaling is a conserved network that regulates embryonic development, organogenesis, tis - sue homeostasis, stem-cell function, differentiation, migration, and tissue polarity8–11. In the canonical pathway, β-catenin stabilization and nuclear translocation activate TCF/LEF-dependent transcriptional programs that influ- ence cell fate and stromal-cell behavior. In endometriosis, dysregulated Wnt/β-catenin activity has been linked to fibrotic remodeling through the regulation of connective tissue growth factor, collagen I, α-smooth muscle actin, and fibronectin12. Other disease-related regulators, including FOXP1 and WEE1, have also been reported to promote fibrotic changes in endometriotic stromal cells through Wnt/β-catenin-associated mechanisms13,14. Inflammation is another central feature of the endometriotic microenvironment. A broad range of inflammatory mediators, including IL-1β, IL-17 A, IL-6, IL-8, IL-10, TNF-α, and VEGF, has been implicated in endometrio - sis15. In particular, increased IL-6 and IL-8 concentrations have been reported in the peritoneal fluid of women with active endometriosis16,17. IL-6 and IL-8 are of particular relevance because they are produced not only by immune cells but also by endometrial and endometriotic cells 15,18. These mediators contribute to angiogenesis, immune-cell recruitment, adhesion, proliferation-associated responses, and lesion progression 19–24. Therefore, stromal-cell behavior, inflammatory signaling, and tissue remodeling are closely connected processes in the biol- ogy of endometriosis. Current treatment strategies include hormonal suppression and surgery. Although these approaches are useful for many patients, hormonal therapy may not be suitable for women seeking fertility preservation, and recurrence or persistence of symptoms remains a major clinical problem25,26. These limitations have encouraged the evalu- ation of non-hormonal experimental approaches that target disease-relevant cellular pathways. Because aberrant Wnt/β-catenin signaling has been implicated in lesion growth, fibrosis, and invasive behavior, this pathway rep- resents a relevant target for investigating new pharmacological strategies in endometriosis research12,27,28. Salinomycin is a natural polyether carboxylic ionophore isolated from Streptomyces albus and has long been used in veterinary medicine 29. Beyond this use, it has been studied for its anti-tumor and pathway-modulat - ing properties in several experimental models 29,30. Previous studies have shown that salinomycin can attenuate Wnt/β-catenin signaling, including inhibition of Wnt-induced LRP6 phosphorylation and reduction of LRP6- associated signaling31,32. It has also been associated with DNA-damage responses, reactive oxygen species gener- ation, mitochondrial membrane depolarization, caspase-3 activation, PARP cleavage, oxidative stress, autophagy, modulation of growth and migration, and p38 MAPK activation 33–39. These reported activities suggest that sali- nomycin may be useful for examining Wnt-related, inflammatory, and survival-associated responses in endome- triotic stromal-cell models. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-67394-0 Based on this rationale, the present study investigated the effects of salinomycin on primary endometriotic stromal cell cultures derived from ectopic lesions and on primary control endometrial stromal cell cultures. We evaluated MTT-derived viable-cell/metabolic activity, invasive behavior, IL-6 and IL-8 secretion, β-catenin phosphorylation at Ser45, and the expression of AXIN2, Cyclin D1, MMP-9, and Caspase-3. The aim was to characterize in vitro cellular responses associated with salinomycin exposure and Wnt/β-catenin signaling, while avoiding overinterpretation of the findings as direct evidence of clinical therapeutic efficacy.

Sample collection This study was approved by the Research Ethics Committee of Urmia University of Medical Sciences (approval no. IR.UMSU.HIMAM.REC.1401.057). All experiments were conducted in accordance with relevant ethical guidelines and regulations, including the Declaration of Helsinki. Written informed consent was obtained from all participants before sample collection. Ectopic endometrial biopsies were collected from 10 women with ovar- ian endometriosis (aged 29–43 years) who underwent laparoscopic surgery. Endometrial tissues were also col - lected from 10 women in the control group who had benign gynecological disorders, were confirmed to be free of endometriosis by laparoscopic evaluation, and underwent the same surgical procedure. Control biopsies were obtained from the functional layer at several sites in the fundal region of the uterine cavity using a biopsy catheter (Pipelle biopsy, Behrad Rouyesh Royan, Iran). All participants had regular menstrual cycles, had not used hor - monal medications for at least 3 months before surgery, and had no history of malignant or autoimmune disease. All biopsy samples were collected during the early secretory phase of the menstrual cycle (days 16–19), deter - mined from the date of the last menstrual period and confirmed by histopathological evaluation. All patients with endometriosis had stage III disease according to the American Society for Reproductive Medicine classification. Samples were immediately placed in Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F12) containing 1% penicillin–streptomycin and transported to the laboratory on ice. Cell isolation and culture Primary endometriotic and control endometrial stromal cell cultures were established according to the protocol described by Karamian et al.40 Briefly, ectopic endometriotic tissue samples from women with ovarian endome- trioma and control endometrial biopsies from women without endometriosis were minced into small fragments and digested with 0.1% collagenase I (Gibco, New York, NY , USA) at 37 °C for 1 h. The digested suspension was sequentially filtered through 70-µm and 40-µm cell strainers to reduce undigested tissue fragments and glan- dular epithelial components. The resulting single-cell suspension was seeded into T25 culture flasks containing DMEM/F12 medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (Gibco, Grand Island, NY , USA) and incubated at 37 °C in a humidified atmosphere with 5% CO₂. After initial attachment, non-adherent cells and debris were removed by medium replacement, and adherent fibroblast-like cells were expanded for subsequent experiments. Primary cultures were established separately from individual participants and were not pooled. Although tissue samples were collected from 10 participants in each group, cultures from three independent donors per group were used for the reported in vitro experiments. Each donor-derived culture constituted one biological replicate ( n = 3 per group). Throughout the manuscript, the terms “primary endome - triotic stromal cell cultures” and “primary control endometrial stromal cell cultures” are used as operational designations based on tissue source, the isolation procedure, adherent fibroblast-like morphology, and Vimentin expression. These terms should not be interpreted as evidence of complete lineage purity. Vimentin and CK18 immunophenotyping was used to characterize the cultures as described below. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-67394-0 Flow cytometric analysis of vimentin and CK18 expression Vimentin and CK18 expression was examined by flow cytometry to characterize the primary cell cultures. Vimen- tin was used as a stromal/mesenchymal-associated marker, whereas CK18 served as an epithelial-associated marker. Cells were fixed with 4% paraformaldehyde and washed once with phosphate-buffered saline (PBS; Sigma-Aldrich, P4417). To minimize nonspecific antibody binding, the cells were blocked with 10% (v/v) goat serum (Sigma-Aldrich, G9023) prepared in PBS for 30 min at room temperature. After removal of the blocking solution, the cells were incubated with the corresponding primary antibodies, diluted 1:100 in PBS, for 4 h at 37 °C. The cells were then washed once with PBS and incubated with the appropriate FITC-conjugated secondary antibodies at a dilution of 1:150 for 45 min at 37 °C in the dark. Following incubation, the samples were washed, centrifuged, resuspended in PBS, and analyzed using a BD FACSCalibur flow cytometer. Vimentin and CK18 were evaluated in separate tubes, and FITC-positive events were defined relative to the corresponding unstained controls. Because Vimentin and CK18 were analyzed in separate tubes, their co-expression within individual cells and the relative proportions of distinct cell populations could not be determined. Therefore, the Vimentin/ CK18 profile was used only for descriptive characterization of the cultures and was not considered sufficient to establish complete lineage purity. The absence of additional stromal-cell markers, such as CD10, ER, and HOXA10, is acknowledged as a limitation of the study. MTT-based viable-cell/metabolic activity assessment The effects of salinomycin on MTT-derived viable-cell/metabolic activity were evaluated using the MTT colo - rimetric assay. Primary endometriotic stromal cell cultures and primary control endometrial stromal cell cul - tures were seeded into 96-well plates at a density of 5 × 10³ cells per well in 100 µL of DMEM/F12 medium supplemented with 10% fetal bovine serum and antibiotics. After overnight attachment, cells were treated with salinomycin at 0.01, 0.1, 1, or 10 µM for 24, 48, or 72 h. Salinomycin was freshly prepared in DMSO, and the final DMSO concentration did not exceed 0.1%. Vehicle-treated cells received DMSO alone, and untreated cells served as negative controls. At each time point, 10 µL of MTT solution (5 mg/mL in PBS) was added to each well, followed by incubation for 3–4 h at 37 °C. The medium was then removed, and 100 µL of DMSO was added to dissolve the formazan crystals. Absorbance was measured at 570 nm with a reference wavelength of 630 nm using a microplate reader. The MTT signal was expressed as a percentage of the vehicle-treated control group. Experiments were performed using primary cultures derived from three independent donors per group, with each donor-derived culture representing one biological replicate ( n = 3 per group). Each donor-derived culture was analyzed in technical triplicate, and the technical replicate values were averaged to generate one value per donor before statistical analysis. IC₅₀ values were calculated by nonlinear regression analysis of dose–response curves using GraphPad Prism version 6.0. For the endpoint MTT-based viable-cell activity assay, cells were seeded at 1 × 10⁴ cells per well and treated under the selected experimental condition. MTT staining and absorbance measurement were performed as described above, without repeating the full protocol. Because MTT reduction reflects cellular metabolic activity and is influenced by assay conditions in addition to cell number, the results were interpreted as changes in viable-cell/metabolic signal rather than as direct evidence of proliferation or cyto- toxicity41. The 24-, 48-, and 72-h measurements represent a time-course MTT response and not a direct prolifera- tion curve. Proliferation-associated effects were further evaluated by Cyclin D1 expression analysis. This assay was performed based on the previously described protocol with minor modifications40. Cell invasion assay The invasive behavior of stromal cells was assessed using Matrigel-coated Transwell inserts with an 8-µm pore membrane. Briefly, cells were collected by trypsinization, washed with phosphate-buffered saline (PBS), centrifuged, and resuspended in a medium containing reduced serum. After determining cell numbers using a Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-67394-0 hemocytometer, 1 × 10⁵ cells were placed into the upper compartment in DMEM/F12 supplemented with 2% fetal bovine serum. The lower compartment was filled with complete culture medium to create a chemotactic gradi - ent. Cells were then incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO₂. During the 24-h invasion assay, cells were left untreated or exposed to vehicle or 1.1 µM salinomycin. At the end of the incubation period, non-invasive cells remaining on the upper side of the membrane were care- fully removed using a sterile cotton swab. Cells that had migrated to the underside of the membrane were fixed and stained with 0.1% Giemsa. Invasive cells were quantified by counting five randomly selected microscopic fields per insert using a light microscope at 200× magnification. Data were expressed as the mean number of invaded cells per field for each experimental group, as previously described40. ELISA-based quantification of cytokines and phosphorylated β-catenin Levels of phosphorylated β-catenin at Ser45 were determined using the InstantOne™ ELISA kit (eBioscience, USA). Cells were seeded into 96-well plates at a density of 3 × 10⁴ cells per well and allowed to adhere overnight. Cells were subsequently lysed with 100 µL of 1× lysis buffer, and 50 µL of each cell lysate, together with the kit- provided controls, was transferred into ELISA wells in triplicate. An equal volume (50 µL) of Antibody Cocktail was then added, and plates were incubated for 1 h at room temperature with gentle agitation. After three washing steps, 100 µL of detection reagent was applied for 20 min, followed by termination of the reaction with 100 µL of stop solution. Optical density was measured at 450 nm. Concentrations of secreted IL-6 and IL-8 in conditioned culture media were quantified using DuoSet® ELISA kits (R&D Systems, USA) in accordance with the manufacturer’s protocols. The assay sensitivities were 9.4 pg/ mL for IL-6 and 31.2 pg/mL for IL-8. Total protein content in cell lysates was measured using the Bradford pro- tein assay (Bio-Rad) and used for data normalization. The cytokine-assay procedures were based on Karamian et al. with minor modifications42. For each group, assays were performed using three independent donor-derived cultures (n = 3 biological replicates). Each donor-derived culture was analyzed in technical triplicate, and techni- cal replicate values were averaged before statistical analysis. RNA extraction and quantitative real-time PCR Total RNA was isolated and purified from cultured cells using RiboEx reagent (GeneAll, Seoul, Korea). Comple- mentary DNA (cDNA) was synthesized from 500 ng of RNA using a cDNA synthesis kit (Qiagen, Hilden, Ger- many). Quantitative real-time PCR (qPCR) was performed in technical triplicate for each donor-derived culture using gene-specific primers and SYBR ® Green Master Mix (Ampliqon, Odense, Denmark) on a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Gene-expression levels were normal - ized to GAPDH as the internal reference. Primer sequences are summarized in Table 1. The thermal cycling protocol included an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 40 s for combined annealing and extension. Relative expression levels of AXIN2, Caspase-3, Cyclin D1, and MMP-9 were calculated using the 2 − ΔΔCt method with REST-RG software (version 3). The resulting Gene Sense primers Antisense primers Cyclin D1 5 ´ - A T T A G G T T C C A T C C T T T A C G T G - 3 ´5 ´ - G T A C A G A G A T G C C T A G A A C C C - 3 ´ Caspase-3 111 5 ´ - G G A A G C G A A T C A A T G G A C T C T G G − 3´ 5 ´ - G C A T C G A C A T C T G T A C C A G A C C − 3´ MMP-9 5 ´ - C A G G C A G C T G G C A G A G G A A T - 3 ´ 5 ´ - T T C G A C T C T C C A C G C A T C T C - 3 ´ Axin-2 5 ´ - T G A G C G G G A T G C T T T G A A C - 3 ´ 5 ´ - A T C C T G T C T C T G T G C A T T G C T G - 3 ´ GAPDH 5 ’ - C G C T T C G G C A G C A C A T A T A C - 3 ’ 5 ’ - A A A T A T G G A A C G C T T C A C G A - 3 ’ Table 1 Sequences of the primers used for mRNA quantitation by real-time RT-PCR. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-67394-0 donor-level relative-expression values were used for the statistical analyses described below. A no-template con- trol reaction without cDNA was included for each gene42. Statistical analyses Data are presented as mean ± standard deviation (SD), as indicated in the corresponding figure legends. The indi- vidual donor-derived primary culture was considered the biological unit of analysis. For each assay, technical replicate measurements were averaged to generate a single value for each donor and were not treated as inde - pendent observations. Statistical analyses were therefore based on three independent donor-derived cultures per group (n = 3 biological replicates). For the dose- and time-response MTT experiment, the effects of salinomycin concentration and exposure time were evaluated using two-way analysis of variance (ANOV A), followed by Tukey’s multiple-comparisons test. For the endpoint MTT assay, cell-invasion assay, cytokine measurements, phospho-β-catenin ELISA, and gene-expression analyses, differences among experimental groups were evalu - ated using one-way ANOV A followed by Tukey’s post hoc test. Statistical analyses were performed using SPSS software, version 19.0 (IBM Corp., Armonk, NY , USA). A p value < 0.05 was considered statistically significant.

Characterization of isolated primary cell cultures Flow cytometry was used to assess Vimentin and CK18 expression in the isolated primary cell cultures. Vimentin was examined as a stromal/mesenchymal-associated marker, whereas CK18 was examined as an epithelial-asso- ciated marker. Vimentin staining showed 85.0% FITC-positive events compared with 3.85% in the corresponding control (Fig. 1a–c). CK18 staining showed 79.6% FITC-positive events compared with 5.61% in the correspond- ing control (Fig. 1d–f). Because Vimentin and CK18 were assessed in separate tubes, these findings do not distin- guish marker co-expression within the same cells from the presence of distinct cell populations. Thus, the marker profile supported the operational designation of the cultures as primary endometriotic and control endometrial stromal cell cultures but did not establish complete lineage purity. Salinomycin reduced MTT-derived viable-cell/metabolic activity A time-course analysis of salinomycin effects on MTT-derived viable-cell/metabolic activity was performed in primary endometriotic stromal cell cultures using concentrations of 0.01, 0.1, 1, and 10 µM over 24, 48, and 72 h. Salinomycin produced a progressive reduction in the MTT signal with increasing concentration and exposure time, with the strongest reduction observed after 72 h (Fig. 2). Based on the dose- and time-dependent response analysis, 1.1 µM salinomycin was selected for subsequent endpoint experiments. These measurements were not interpreted as a direct proliferation curve. The endpoint MTT assay was then used to compare primary control endometrial and endometriotic stromal cell cultures under baseline and treatment conditions. Baseline MTT absorbance was lower in endometriotic stromal cell cultures than in control endometrial stromal cell cultures (Fig. 3A). Because the MTT assay mainly reflects viable-cell metabolic activity, this difference was interpreted as a change in MTT-derived signal rather than as direct evidence of altered proliferation. In primary endometriotic stromal cell cultures, treatment with 1.1 µM salinomycin for 72 h markedly reduced the MTT-derived signal compared with vehicle-treated cells (Fig. 3B). A smaller but statistically significant reduction was also observed in primary control endometrial stro- mal cell cultures (Fig. 3C). Overall, these findings show that salinomycin reduced viable-cell/metabolic activity, with a stronger effect in endometriotic stromal cell cultures. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-67394-0 Salinomycin reduced invasive behavior in primary stromal cell cultures The invasive behavior of primary control endometrial and endometriotic stromal cell cultures was evaluated using Matrigel-coated Transwell inserts. Representative images showed invaded cells under control, vehicle- treated, and salinomycin-treated conditions (Fig. 4). Quantification was performed after the 24-h Transwell inva- sion assay by counting invaded cells in randomly selected microscopic fields. At baseline, endometriotic stromal cell cultures showed a higher number of invaded cells per field than control endometrial stromal cell cultures (Fig. 5A). Exposure to 1.1 µM salinomycin reduced invasion in primary control endometrial stromal cell cultures compared with vehicle-treated cells (Fig. 5B). The reduction was more pronounced in primary endometriotic stromal cell cultures (Fig. 5C). These results indicate that salinomycin attenuated invasive behavior under the experimental conditions, particularly in endometriotic stromal cell cultures. Salinomycin increased phospho-β-catenin Ser45 signal in endometriotic stromal cell cultures Phospho-β-catenin at Ser45 was measured by ELISA and reported as optical density values. At baseline, primary endometriotic stromal cell cultures showed a lower phospho-β-catenin Ser45 signal than primary control endo - metrial stromal cell cultures (Fig. 6A). Treatment with 1.1 µM salinomycin for 72 h increased the phospho-β- catenin Ser45 signal in primary endometriotic stromal cell cultures compared with vehicle-treated cells (Fig. 6B). In contrast, salinomycin did not produce a significant change in primary control endometrial stromal cell cultures (Fig. 6C). These data suggest that salinomycin was associated with altered β-catenin regulation, mainly in endo- metriotic stromal cell cultures. Fig. 1 Flow-cytometric characterization of isolated primary cell cultures. Vimentin (a–c) and CK18 (d–f) were evaluated as stromal/mesenchymal-associated and epithelial-associated markers, respectively. Vimentin staining yielded 85.0% FITC- positive events versus 3.85% in the corresponding control, whereas CK18 staining yielded 79.6% versus 5.61%. Because Vimentin and CK18 were assessed in separate tubes, these data do not establish single-cell co-expression or complete lineage purity. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-67394-0 Salinomycin reduced IL-6 and IL-8 secretion Secreted IL-6 and IL-8 levels were measured in conditioned culture media by ELISA and normalized to total protein content. Baseline IL-6 secretion was comparable between untreated primary control endometrial stromal cell cultures and primary endometriotic stromal cell cultures from the same experimental set (Fig. 7A). After treatment with 1.1 µM salinomycin for 72 h, IL-6 secretion was reduced in primary control endometrial stromal cell cultures compared with vehicle-treated cells (Fig. 7B). A stronger reduction was observed in primary endo- metriotic stromal cell cultures (Fig. 7C). For IL-8, the initial baseline comparison showed higher secretion in primary endometriotic stromal cell cultures than in primary control endometrial stromal cell cultures (Fig. 8A). Under endpoint treatment conditions, salinomycin produced a modest reduction in IL-8 secretion in primary control endometrial stromal cell cultures (Fig. 8B). In primary endometriotic stromal cell cultures, salinomycin markedly reduced IL-8 secretion compared with vehicle-treated cells (Fig. 8C). Together, these findings indicate that salinomycin reduced pro-inflammatory cytokine secretion, with a more evident effect in endometriotic stro- mal cell cultures. Salinomycin altered expression of proliferation-, invasion-, and apoptosis-associated markers Cyclin D1 mRNA expression was evaluated as a proliferation-associated marker. Baseline Cyclin D1 expression did not differ significantly between primary control endometrial stromal cell cultures and primary endometriotic Fig. 2 Dose- and time-dependent effects of salinomycin on MTT-derived viable-cell/metabolic activity in primary endo - metriotic stromal cell cultures. Cells were exposed to 0.01, 0.1, 1, or 10 µM salinomycin for 24, 48, or 72 h. MTT signal is expressed as a percentage of the vehicle-treated control and represents viable-cell metabolic activity rather than a direct measure of proliferation. Data are mean ± SD from three independent donor-derived cultures ( n = 3 biological replicates). Each culture was measured in technical triplicate, and replicate wells were averaged before analysis. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-67394-0 stromal cell cultures (Fig. 9A). Salinomycin treatment did not significantly alter Cyclin D1 expression in primary control endometrial stromal cell cultures (Fig. 9B). In contrast, treatment with 1.1 µM salinomycin for 72 h sig- nificantly reduced Cyclin D1 mRNA expression in primary endometriotic stromal cell cultures compared with vehicle-treated cells (Fig. 9C). These results provide marker-based support for reduced proliferation-associated signaling in endometriotic stromal cell cultures. MMP-9 mRNA expression was assessed as an invasion-associ - ated marker. At baseline, MMP-9 expression did not show a statistically significant difference between primary control endometrial and endometriotic stromal cell cultures (Fig. 10A). Salinomycin did not significantly change MMP-9 expression in primary control endometrial stromal cell cultures (Fig. 10B). However, in primary endo- metriotic stromal cell cultures, salinomycin significantly reduced MMP-9 mRNA expression compared with vehicle-treated cells (Fig. 10C). This decrease in MMP-9 expression was consistent with the reduced invasive behavior observed in the Transwell assay. Caspase-3 mRNA expression was measured as an apoptosis-associated marker. Baseline Caspase-3 expression was significantly lower in primary endometriotic stromal cell cultures than in primary control endometrial stromal cell cultures (Fig. 11A). Salinomycin did not significantly alter Fig. 3 Effects of salinomycin on MTT-derived viable-cell/metabolic activity in primary control endometrial and endome - triotic stromal cell cultures. (A) Baseline MTT absorbance in control endometrial and endometriotic cultures. ( B, C) MTT absorbance after treatment with vehicle or 1.1 µM salinomycin for 72 h in endometriotic and control endometrial cultures, respectively. Absorbance was measured at 570 nm. MTT is a viable-cell metabolic readout and does not directly distinguish proliferation inhibition from cytotoxicity. Data are mean ± SD from three independent donor-derived cultures per group (n = 3 biological replicates). Each culture was measured in technical triplicate, and replicate wells were averaged before analysis. *p < 0.05; ***p < 0.001. Normal, primary control endometrial stromal cell culture; Ectopic, primary endometriotic stromal cell culture; CESC, primary control endometrial stromal cell culture; EESC, primary endometriotic stromal cell culture; SAL, salinomycin. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-67394-0 Caspase-3 expression in primary control endometrial stromal cell cultures (Fig. 11B). In primary endometriotic stromal cell cultures, treatment with 1.1 µM salinomycin for 72 h significantly increased Caspase-3 mRNA expression compared with control and vehicle-treated cells (Fig. 11C). These findings suggest that the reduction in MTT-derived viable-cell/metabolic signal may partly reflect apoptosis-associated transcriptional responses in endometriotic stromal cell cultures. Salinomycin reduced AXIN2 expression AXIN2 mRNA expression was assessed as a Wnt/β-catenin pathway-associated transcriptional marker. At base- line, AXIN2 expression was higher in primary endometriotic stromal cell cultures than in primary control endo- metrial stromal cell cultures (Fig. 12A). Treatment with 1.1 µM salinomycin for 72 h significantly reduced AXIN2 mRNA expression in primary control endometrial stromal cell cultures (Fig. 12B). A more pronounced reduction was observed in primary endometriotic stromal cell cultures (Fig. 12C). Together with the increase in phospho-β-catenin Ser45 signal, the reduction in AXIN2 expression is consistent with attenuation of Wnt/β- catenin pathway-associated transcriptional activity following salinomycin exposure, particularly in primary endometriotic stromal cell cultures. Fig. 4 Representative images of Matrigel Transwell invasion assays in primary control endometrial and endometriotic stro- mal cell cultures. CESCs (top row) and EESCs (bottom row) are shown under control, vehicle-treated, and 1.1 µM salinomy- cin-treated conditions during the 24-h assay. Invaded cells on the lower membrane surface were fixed, stained with Giemsa, and imaged at 200× magnification. Quantification is shown in Fig. 5. Scale bar, 100 μm. CESC, primary control endometrial stromal cell culture; EESC, primary endometriotic stromal cell culture; SAL, salinomycin. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-67394-0

Endometriosis is increasingly viewed as a chronic inflammatory disorder in which ectopic endometrial-like lesions acquire abnormal stromal-cell behaviors, including enhanced survival, invasive capacity, tissue remodel- ing, and sustained inflammatory activity 43–45. Although the disease is benign, several of these cellular features overlap with tumor-like biological processes, particularly adhesion, invasion, and resistance to apoptosis 3,44,45. Developmental signaling pathways, including Wnt/β-catenin signaling, have been implicated in these disease- associated phenotypes and may contribute to lesion persistence, fibrotic remodeling, and altered stromal-cell function28,45. Because current hormone-based treatments may not be suitable for all patients, particularly those seeking fertility preservation, there remains a need to investigate non-hormonal experimental approaches that target disease-relevant cellular pathways46. In the present study, we examined the effects of salinomycin in primary control endometrial stromal cell cul - tures and primary endometriotic stromal cell cultures. These terms are retained as operational designations based on tissue source, isolation procedure, adherent fibroblast-like morphology, and Vimentin expression. However, the substantial CK18 signal and separate-tube analysis preclude claims of complete lineage purity or single-cell Vimentin/CK18 co-expression. Within this experimental framework, salinomycin reduced MTT-derived viable- cell/metabolic activity, invasive behavior, inflammatory cytokine secretion, and selected molecular markers Fig. 5 Effects of salinomycin on invasion by primary control endometrial and endometriotic stromal cell cultures. (A) Base- line invasion in control endometrial and endometriotic cultures. (B, C) Invasion after exposure to vehicle or 1.1 µM salino- mycin in control endometrial and endometriotic cultures, respectively, during the 24-h assay. Invaded cells were counted in five randomly selected fields per insert. Data are mean ± SD from three independent donor-derived cultures per group (n = 3 biological replicates). Each culture was analyzed in technical triplicate, and replicate measurements were averaged before analysis. *p < 0.05; **p < 0.01. Normal, primary control endometrial stromal cell culture; Ectopic, primary endometriotic stromal cell culture; CESC, primary control endometrial stromal cell culture; EESC, primary endometriotic stromal cell culture; SAL, salinomycin. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-67394-0 associated with proliferation, invasion, apoptosis, and Wnt/β-catenin-related signaling. These effects were gener- ally more pronounced in primary endometriotic cultures than in control endometrial cultures. The reduction in MTT-derived signal should be interpreted carefully. The MTT assay primarily reflects cel - lular metabolic activity and does not, by itself, distinguish reduced proliferation from reduced metabolic activity, reduced cell number, or cell death41. In the present study, baseline MTT absorbance was lower in endometriotic stromal cell cultures than in control cultures, and salinomycin produced a stronger reduction in MTT-derived Fig. 7 Effects of salinomycin on IL-6 secretion by primary control endometrial and endometriotic stromal cell cultures. IL-6 in conditioned media was quantified by ELISA and normalized to total protein in the corresponding cell lysates. ( A) Base- line IL-6 secretion in untreated control endometrial and endometriotic cultures. ( B, C) IL-6 secretion after treatment with vehicle or 1.1 µM salinomycin for 72 h in control endometrial and endometriotic cultures, respectively. Data are mean ± SD from three independent donor-derived cultures per group ( n = 3 biological replicates). Each culture was analyzed in techni - cal triplicate, and replicate measurements were averaged before analysis. **p < 0.01; ***p < 0.001. Normal, primary control endometrial stromal cell culture; Ectopic, primary endometriotic stromal cell culture; CESC, primary control endometrial stromal cell culture; EESC, primary endometriotic stromal cell culture; SAL, salinomycin. Fig. 6 Effects of salinomycin on phospho-β-catenin (Ser45) in primary control endometrial and endometriotic stromal cell cultures. Phospho-β-catenin (Ser45) was measured by ELISA and reported as optical-density values. (A) Baseline phospho- β-catenin (Ser45) signal in control endometrial and endometriotic cultures. (B, C) Signal after treatment with vehicle or 1.1 µM salinomycin for 72 h in endometriotic and control endometrial cultures, respectively. Data are mean ± SD from three independent donor-derived cultures per group ( n = 3 biological replicates). Each culture was analyzed in technical tripli - cate, and replicate measurements were averaged before analysis. *p < 0.05; **p < 0.01. Normal, primary control endometrial stromal cell culture; Ectopic, primary endometriotic stromal cell culture; CESC, primary control endometrial stromal cell culture; EESC, primary endometriotic stromal cell culture; SAL, salinomycin. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-67394-0 signal in endometriotic stromal cell cultures. This finding suggests differential responsiveness to salinomycin under the present in vitro conditions, but it should not be interpreted as direct evidence of reduced proliferation alone. The observed decrease in Cyclin D1 mRNA expression in salinomycin-treated endometriotic stromal cell cultures provides marker-based support for a reduction in proliferation-associated signaling. Reduced prolif - eration-, migration-, and invasion-related responses have been reported after pharmacological disruption of the TCF/β-catenin complex in endometrial and endometriotic cell models, supporting the relevance of this pathway Fig. 9 Effects of salinomycin on Cyclin D1 mRNA expression in primary control endometrial and endometriotic stromal cell cultures. Relative Cyclin D1 mRNA expression was quantified by real-time PCR and normalized to GAPDH; Cyclin D1 was evaluated as a proliferation-associated marker. ( A) Baseline expression in control endometrial and endometriotic cultures. (B, C) Expression after treatment with vehicle or 1.1 µM salinomycin for 72 h in control endometrial and endome- triotic cultures, respectively. Data are mean ± SD from three independent donor-derived cultures per group (n = 3 biological replicates). Each culture was analyzed in technical triplicate, and replicate measurements were averaged before analysis. **p < 0.01 versus vehicle. Normal, primary control endometrial stromal cell culture; Ectopic, primary endometriotic stromal cell culture; CESC, primary control endometrial stromal cell culture; EESC, primary endometriotic stromal cell culture; SAL, salinomycin. Fig. 8 Effects of salinomycin on IL-8 secretion by primary control endometrial and endometriotic stromal cell cultures. IL-8 in conditioned media was quantified by ELISA and normalized to total protein in the corresponding cell lysates. ( A) Base- line IL-8 secretion in untreated control endometrial and endometriotic cultures. ( B, C) IL-8 secretion after treatment with vehicle or 1.1 µM salinomycin for 72 h in control endometrial and endometriotic cultures, respectively. Data are mean ± SD from three independent donor-derived cultures per group ( n = 3 biological replicates). Each culture was analyzed in techni - cal triplicate, and replicate measurements were averaged before analysis. * p < 0.05; ***p < 0.001. Normal, primary control endometrial stromal cell culture; Ectopic, primary endometriotic stromal cell culture; CESC, primary control endometrial stromal cell culture; EESC, primary endometriotic stromal cell culture; SAL, salinomycin. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-67394-0 in regulating disease-associated cellular behavior27. Direct cell counting, Ki67 or PCNA analysis, and BrdU/EdU incorporation would provide more direct evidence of proliferation inhibition and should be considered in future studies. Invasion is a key feature of lesion establishment and tissue remodeling in endometriosis. In the present study, primary endometriotic stromal cell cultures showed higher basal invasive behavior than control endometrial stromal cell cultures in the Transwell assay. Salinomycin reduced invasion in both cell types, with a stronger Fig. 11 Effects of salinomycin on Caspase-3 mRNA expression in primary control endometrial and endometriotic stromal cell cultures. Relative Caspase-3 mRNA expression was quantified by real-time PCR and normalized to GAPDH; Caspase-3 mRNA was evaluated as an apoptosis-associated transcriptional marker. (A) Baseline expression in control endometrial and endometriotic cultures. (B, C) Expression after treatment with vehicle or 1.1 µM salinomycin for 72 h in control endometrial and endometriotic cultures, respectively. Data are mean ± SD from three independent donor-derived cultures per group (n = 3 biological replicates). Each culture was analyzed in technical triplicate, and replicate measurements were averaged before analysis. * p < 0.05; ** p < 0.01; *** p < 0.001, as indicated by comparison brackets. Normal, primary control endometrial stromal cell culture; Ectopic, primary endometriotic stromal cell culture; CESC, primary control endometrial stromal cell culture; EESC, primary endometriotic stromal cell culture; SAL, salinomycin. Fig. 10 Effects of salinomycin on MMP-9 mRNA expression in primary control endometrial and endometriotic stromal cell cultures. Relative MMP-9 mRNA expression was quantified by real-time PCR and normalized to GAPDH. ( A) Baseline expression in control endometrial and endometriotic cultures. ( B, C) Expression after treatment with vehicle or 1.1 µM salinomycin for 72 h in control endometrial and endometriotic cultures, respectively. Data are mean ± SD from three inde- pendent donor-derived cultures per group ( n = 3 biological replicates). Each culture was analyzed in technical triplicate, and replicate measurements were averaged before analysis. * p < 0.05 versus vehicle. Normal, primary control endometrial stromal cell culture; Ectopic, primary endometriotic stromal cell culture; CESC, primary control endometrial stromal cell culture; EESC, primary endometriotic stromal cell culture; SAL, salinomycin. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-67394-0 reduction in endometriotic stromal cell cultures. This functional change was accompanied by reduced MMP-9 mRNA expression in salinomycin-treated endometriotic stromal cell cultures. Because MMP-9 contributes to extracellular matrix degradation and stromal invasiveness in endometriosis, its downregulation is consistent with the observed reduction in invasion47. However, baseline MMP-9 expression may vary according to tissue origin, disease context, and menstrual-cycle phase 47,48. Therefore, the present findings are best interpreted as show - ing that salinomycin exposure was associated with reduced invasive behavior and decreased expression of an invasion-associated marker, rather than establishing MMP-9 as the sole mediator of the anti-invasive response. The Wnt/β-catenin pathway has been implicated in endometriosis pathogenesis and in the regulation of stro - mal-cell survival, invasion, and fibrosis-related responses 28. In this study, primary endometriotic stromal cell cultures showed lower basal phospho-β-catenin Ser45 signal and higher AXIN2 mRNA expression than con - trol cultures. Phosphorylation of β-catenin at Ser45 creates a priming site for subsequent GSK-3β-dependent phosphorylation and degradation, whereas AXIN2 is a Wnt-responsive transcriptional target that functions as a negative-feedback regulator49,50. Salinomycin increased phospho-β-catenin Ser45 signal and reduced AXIN2 expression, particularly in endometriotic stromal cell cultures. Together, these changes support an association between salinomycin exposure and attenuation of Wnt/β-catenin pathway-associated transcriptional activity. Nevertheless, because the study did not include Wnt reporter assays, pathway rescue experiments, or targeted gain/loss-of-function approaches, these data should be interpreted as pathway-associated evidence rather than definitive proof of causal Wnt inhibition. AXIN2 can also have context-dependent functions; in colorectal cancer models, elevated AXIN2 promoted a Snail1-dependent epithelial-mesenchymal transition program and invasive/ metastatic behavior51. Therefore, the reduction in AXIN2 after salinomycin exposure provides molecular support for altered Wnt-associated signaling but does not establish a cell-specific causal mechanism in endometriosis. Inflammation is central to the endometriotic microenvironment. IL-6 and IL-8 contribute to immune-cell recruitment, angiogenesis, adhesion, stromal-cell activation, and lesion progression 15,18–24. In the present data - set, baseline IL-6 secretion was comparable between control and endometriotic stromal cell cultures, whereas baseline IL-8 secretion was higher in endometriotic stromal cell cultures. Salinomycin reduced both IL-6 and IL-8 secretion, with a more pronounced reduction in endometriotic stromal cell cultures. These findings sug - gest that salinomycin may modulate inflammatory output in primary stromal-cell cultures. This observation Fig. 12 Effects of salinomycin on AXIN2 mRNA expression in primary control endometrial and endometriotic stromal cell cultures. Relative AXIN2 mRNA expression was quantified by real-time PCR and normalized to GAPDH. ( A) Baseline expression in control endometrial and endometriotic cultures. ( B, C) Expression after treatment with vehicle or 1.1 µM salinomycin for 72 h in control endometrial and endometriotic cultures, respectively. Data are mean ± SD from three inde- pendent donor-derived cultures per group (n = 3 biological replicates). Each culture was analyzed in technical triplicate, and replicate measurements were averaged before analysis. * p < 0.05, as indicated by comparison brackets. Normal, primary control endometrial stromal cell culture; Ectopic, primary endometriotic stromal cell culture; CESC, primary control endo - metrial stromal cell culture; EESC, primary endometriotic stromal cell culture; SAL, salinomycin. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-67394-0 is compatible with reported crosstalk between Wnt/β-catenin signaling and inflammatory pathways, including NF-κB-dependent responses, although the present study did not directly test this interaction52. Resistance to apoptosis is another important feature of ectopic endometrial stromal cells 28,45,53. Consistent with this concept, basal Caspase-3 mRNA expression was lower in primary endometriotic stromal cell cultures than in primary control endometrial stromal cell cultures. Salinomycin increased Caspase-3 mRNA expression in endometriotic stromal cell cultures, whereas no significant change was observed in control cultures. This finding suggests activation of apoptosis-associated transcriptional responses after salinomycin exposure. Importantly, because apoptosis was evaluated only at the mRNA level, these data cannot be taken as definitive evidence of functional apoptosis. Confirmation by protein-level or functional assays, such as cleaved caspase-3 detection, caspase activity assays, Annexin V/PI staining, or TUNEL analysis, would be required in future studies. Pro- apoptotic effects of salinomycin have been demonstrated in cells with dysregulated Wnt signaling 31, supporting a possible association between Wnt-pathway status and salinomycin responsiveness. When compared with previous studies targeting Wnt/β-catenin signaling in endometriosis, our findings are directionally consistent but mechanistically less definitive. Matsuzaki and Darcha showed that pharmacological disruption of the Tcf/β-catenin complex using PKF 115–584 reduced proliferation-, migration-, and invasion- related responses in endometrial and endometriotic epithelial and stromal cell models, with additional effects on MMP-2/MMP-9 activity 27. In a related study, the same group reported that targeting Wnt/β-catenin signal - ing reduced fibrosis-associated markers and collagen gel contraction in endometrial and endometriotic stromal cells, and also affected fibrosis progression in a xenograft model of endometriosis12. Our data extend this general concept by showing that salinomycin exposure was accompanied by reduced invasive behavior, lower MMP-9 expression, increased β-catenin Ser45 phosphorylation, and decreased AXIN2 expression in primary endometri- otic stromal cell cultures. However, unlike studies using pathway-specific antagonists, β-catenin knockdown, or in vivo models, the present study relied on pathway-associated markers rather than direct functional interrogation of Wnt activity. Therefore, the observed molecular changes should be interpreted as being consistent with attenu- ation of Wnt/β-catenin-associated transcriptional activity, rather than as conclusive evidence of causal pathway inhibition. The salinomycin-related findings should also be considered in light of studies performed in malignant gyne - cological and non-gynecological models. Salinomycin has been reported to inhibit Wnt signaling by blocking Wnt-induced LRP6 phosphorylation and promoting LRP6 downregulation, and additional studies have shown suppression of LRP6-associated Wnt/β-catenin signaling in breast and prostate cancer cells31,32. In gynecological malignancy models, salinomycin reduced proliferation, migration, and invasion in human endometrial cancer stem-like cells and repressed epithelial-mesenchymal transition-associated responses in epithelial ovarian cancer cells through Wnt/β-catenin-related mechanisms54,55. These reports are broadly compatible with our observations in primary endometriotic stromal cell cultures, particularly the reduction in MTT-derived viable-cell/metabolic activity, invasion, Cyclin D1, MMP-9, and AXIN2, together with increased Caspase-3 mRNA expression. Nev- ertheless, direct extrapolation from malignant models to endometriosis should be avoided. Endometriosis is a benign, hormone-responsive inflammatory disorder, and stromal-cell responses in primary cultures may differ substantially from malignant or cancer stem-like cell systems. Thus, the present findings should be viewed as preclinical, pathway-associated observations that require validation using expanded stromal-cell markers, direct Wnt reporter or rescue experiments, functional apoptosis assays, and in vivo endometriosis models. Several limitations should be considered when interpreting these findings. First, cell characterization was based on the isolation strategy, adherent fibroblast-like morphology, and Vimentin/CK18 profiling. Vimentin and CK18 were detected in separate tubes (85.0% and 79.6% FITC-positive events, respectively), and their co- expression at the single-cell level was not assessed. The substantial CK18 signal may reflect epithelial-cell con- tamination, dual-marker expression, or distinct cell populations; the present data cannot distinguish among these possibilities. Additional markers such as CD10, ER, PR, HOXA10, or IFITM1 were not evaluated. Therefore, the terms “primary endometriotic stromal cell cultures” and “primary control endometrial stromal cell cultures” are operational designations and should not be interpreted as proof of a completely lineage-pure stromal population Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-67394-0 or as identification of multipotent mesenchymal stromal cells. Second, MTT-based assays were used as viable- cell/metabolic readouts and do not directly measure proliferation or distinguish reduced proliferation from altered metabolism or loss of viable cells 41. Direct cell-counting curves, Ki67/PCNA analysis, and BrdU/EdU incor - poration were not performed. Third, Caspase-3 was assessed at the mRNA level only, and functional apoptosis was not directly confirmed. Fourth, the mechanistic interpretation of Wnt/β-catenin involvement was based on phospho-β-catenin Ser45 and AXIN2 expression; direct pathway activity was not tested using reporter or rescue experiments. Finally, the study was conducted in vitro and did not address systemic toxicity, pharmacokinetics, tissue selectivity, or in vivo efficacy. Taken together, the present findings indicate that salinomycin modulates several disease-relevant cellular responses in primary endometriotic stromal cell cultures, including MTT-derived viable-cell/metabolic activity, invasive behavior, inflammatory cytokine secretion, apoptosis-associated gene expression, and Wnt/β-catenin pathway-associated markers. The stronger responses observed in endometriotic stromal cell cultures suggest dif- ferential sensitivity under the present in vitro conditions. These results support further investigation of Salinomy- cin and Wnt-directed pathway modulation as experimental, non-hormonal research approaches in endometriosis biology.

In this study, Salinomycin modulated several disease-relevant cellular responses in primary endometriotic stro - mal cell cultures under in vitro conditions. Salinomycin reduced MTT-derived viable-cell/metabolic activity, invasive behavior, IL-6 and IL-8 secretion, and the expression of the proliferation- and invasion-associated mark- ers Cyclin D1 and MMP-9. These effects were generally more pronounced in primary endometriotic cultures than in primary control endometrial cultures. At the molecular level, Salinomycin increased Phospho-beta-Catenin (Ser45) signal and reduced AXIN2 expression, findings consistent with modulation of Wnt/β-catenin pathway- associated transcriptional activity. Salinomycin also increased Caspase-3 mRNA expression in endometriotic cultures, suggesting an apoptosis-associated transcriptional response. However, because the study was limited to an in vitro model and did not include expanded cell-lineage validation, direct proliferation assays, functional apoptosis assays, Wnt reporter assays, or pathway-specific rescue experiments, the findings should be interpreted as pathway-associated experimental evidence rather than proof of therapeutic efficacy. Further studies using expanded phenotypic validation, functional mechanistic assays, and in vivo endometriosis models are required before cell-specific mechanisms or therapeutic relevance can be established.

The authors gratefully acknowledge the technical and administrative support provided by the Repro - ductive Health Research Center and the Clinical Research Institute of Urmia University of Medical Sciences. We also thank all participants who provided tissue samples for this study. During the preparation of this manuscript, the authors used Chat- GPT to improve language clarity and readability. After using this tool, all authors carefully reviewed, revised, and approved the final content and take full responsibility for the accuracy, integrity, and scientific content of the publication. Author contributions HGB conceptualized and designed the study, performed data curation, and wrote the original draft. FM analyzed the data and reviewed and edited the manuscript. MGB, SS contributed to the investigation and manuscript editing. MP was involved in methodology development, validation, and manuscript editing. All authors have read and ap - proved the final version of the manuscript. Funding No funding was received for conducting this study. Data availability The data used and/or analyzed during thecurrent study are available from the corresponding author on reasonable request. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-67394-0 Declarations Competing interests The authors declare no competing interests. Declaration of generative AI and AI-assisted technologies in the writing process During the preparation of this work, the authors used ChatGPT to improve the language of the manuscript. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 Interna- tional License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Cre- ative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit h t t p : / / c r e a t i v e c o m m o n s . o r g / l i c e n s e s / b y - n c - n d / 4 . 0 / .

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Salinomycin repressed the epithelial–mesenchymal transition of epithelial ovarian cancer cells via downregulating Wnt/β-catenin pathway. OncoTargets Therapy 28, 1317–1325 (2017). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Authors and Affiliations Sonia Sadeghpour1 · Morteza Ghasemnejad-Berenji1 · Farzad Maleki2 · Mohammad Reza Pashaei2 · Hojat Ghasemnejad-Berenji1,3 Hojat Ghasemnejad-Berenji [email protected] 1 Reproductive Health Research Center, Clinical Research Institute, Urmia University of Medical Sciences, Urmia, Iran 2 Patient Safety Research Center, Clinical Research Institute, Urmia University of Medical Sciences, Urmia, Iran 3 Reproductive Biology, Reproductive Health Research Center, Clinical Research Institute, Urmia University of Medical Sciences, Urmia, Iran