BIBR1532 Affects Endometrial Cell Proliferation, Migration, and Invasion in Endometriosis via Telomerase Inhibition and MAPK Signaling

Objectives: The effect of telomerase inhibitor BIBR1532 on endometriotic cells was investigated to explore the inhibitory effect of targeting telomerase on endometriosis. Design: In vitro primary cell culture study. Participants/Materials: Primary endometrial cells derived from eutopic and ectopic endometrium in patients with endometriosis. Setting: The study was conducted in the university hospital. Methods: Paired eutopic and ectopic endometrial cells were collected from 6 patients from January 2018 to July 2021. A TRAP assay was performed to detect the telomerase activity of the cells. MTT, cell cycle, apoptosis, migration, and invasion assays were performed to study the inhibitory effect of BIBR1532. Enrichment analysis was performed to identify the key pathways involved in endometriosis progression and telomerase action. Then, Western blotting was used to investigate the expression of related proteins. Results: BIBR1532 treatment significantly inhibited the growth of eutopic and ectopic endometrial cells, with apoptosis and cell cycle signaling involved. Migration and invasion, important characteristics for the establishment of ectopic lesions, were also inhibited by BIBR1532. The MAPK signaling cascade, related to telomerase and endometriosis, was decreased in eutopic and ectopic endometrial stromal cells with the treatment of BIBR1532. Limitations: The severe side effects of telomerase inhibitors might be the main obstacle to clinical application, so it is necessary to find better drug delivery methods in vivo. Conclusions: The telomerase inhibitor BIBR1532 affects endometrial cell proliferation, migration, and invasion in endometriosis.


Introduction
Endometriosis, commonly defined as the presence of endometrial-like tissue outside of the uterine cavity, is one of the most common reproductive diseases, affecting 10% of women during their reproductive years as well as infertile women with a prevalence of 30%-50% [1,2]. Dysmenorrhea, dyspareunia, chronic pelvic pain, and infertility are common symptoms of patients with endometriosis [1][2][3]. Although there are many treatments for endometriosis, the disease is difficult to cure because both medical therapy and conservative surgery are associated with high recurrence rates, and radical surgery is commonly accompanied by severe complications [4,5], indicating that a more effective target for endometriosis treatment is needed.
Sampson's retrograde menstruation theory is the most widely accepted hypothesis for the occurrence of karger@karger.com www.karger.com/goi endometriosis and postulates that retrograde menstruation enters the cavity, resulting in endometriosis [6]. Compared with women without endometriosis, the prevalence of retrograde menstruation has been reported to be higher in women with endometriosis [7]. However, the prevalence of menstrual reflux in women was reported to be 60%-90%, higher than the incidence of endometriosis, which is 10% [8]. So, there may be particular aberrations of the eutopic endometrium in women with endometriosis that promote the survival of endometrial tissue outside the uterine cavity.
Telomerase is an RNA-dependent DNA polymerase that is involved in normal endometrial regeneration and abnormal endometrial pathologies through telomere maintenance and nontelomeric functions [9]. It is reported that the telomerase activity in the eutopic endometrium during the secretory phase was higher in women with endometriosis than that in healthy women [10][11][12]. Telomerase components, such as telomerase reverse transcriptase (TERT) and telomerase RNA component, were also found to be higher in the eutopic endometrium of endometriosis patients [10,13]. The longer mean endometrial telomere length, which is maintained by telomerase, has been detected in the eutopic endometrium of women with endometriosis [9][10][11][12]. The differentially expressed genes related to telomerase activity in the eutopic endometrium between endometriosis patients and healthy women have also been filtered [13]. These findings imply that telomerase may be the abnormality promoting the ectopic growth of cells and thus a potential treatment target.
BIBR1532 (2-[(E)-3-naphthalen-2-yl-but-2-enoylamino]benzoic acid) is a synthetic nonnucleoside, noncompetitive small-molecule telomerase inhibitor [14,15]. By binding to the hydrophobic cavity on the outer surface of the thumb domain of telomerase, BIBR1532 can inhibit telomerase activity specifically, thereby preventing the elongation of telomere terminal repeats, which can affect cellular functions [14,15]. BIBR1532 is widely used for the study of telomerase function and has been reported to inhibit cell viability effectively across several cancer cell types [16][17][18][19][20]. Our previous study also found that BIBR1532 can inhibit the proliferation of endometrial cancer cell lines by inhibiting their telomerase activity [21]. Therefore, in the present study, we investigated the inhibitory effect of BIBR1532 on eutopic and ectopic endometrial cells derived from endometriotic patients as well as the preliminary clinical value of targeting telomerase for endometriosis treatment.

Clinical Specimens
Ectopic endometriotic and matched eutopic endometrial samples were collected from 6 women with endometriosis combined with grade II-III cervical intraepithelial neoplasia or uterine leiomyoma who underwent surgery at Beijing Obstetrics and Gynecology Hospital from January 2018 to July 2021. None of the patients received any hormone therapy for at least 6 months prior to the surgery, and all the patients had normal regular menstrual cycles and were in the secretory period. The pathological diagnosis was performed preoperatively and confirmed postoperatively. Endometrial biopsies were dated using histological criteria, the date of the last menstrual period, and hormone profiles.
This study protocol was reviewed and approved by the Beijing Obstetrics and Gynecology Hospital affiliated with the Capital Medical University Ethics Committee (approval number 2019-KY-002-01). Written informed consent was obtained from all the participants, and all experiments complied with the guidelines for human studies and conformed to the Declaration of Helsinki.

Isolation and Identification of Primary Endometrial Cells
Primary eutopic and ectopic endometrial cells were isolated according to the method described by Chen [22] with some modifications. Briefly, the fresh tissue samples were cut into l-mm 3 pieces and digested in phosphate-buffered saline (PBS) containing collagenase type I (1 mg/mL, 15 U/mg) and 1% (v/v) penicillin/streptomycin for 1 h in an orbital shaker at 37°C. Tissue residue was removed by filtration through cell strainers with 100µm pores (BD Bioscience, Franklin Lakes, NJ, USA), and then the stromal cells and epithelial cells were separated through strainers with 40-µm pores (BD Bioscience). Human eutopic endometrial epithelial cells (HEuEECs) were collected from the pellet in sieves and cultured in defined keratinocyte serum-free medium (DKSFM; Gibco, Grand Island, NY, USA). The filtrate was centrifuged at 1,200 g for 5 min, and the supernatant was discarded. Then, human eutopic endometrial stromal cells (HEuESCs) and human ectopic endometrial stromal cells (HEcESCs) were suspended and cultured in DMEM/F12 with 10% fetal bovine serum (FBS; Corning, Tewksbury, MA, USA) and 1% (v/v) penicillin/ streptomycin. Immunofluorescent staining was performed to determine the purity of the isolated cells using vimentin (5741, Cell Signaling Technology, Beverly, MA, USA) and pankeratin (4545, Cell Signaling Technology, Beverly, MA, USA) antibodies for stromal cells and epithelial cells, respectively. The purity of the isolated stromal cells = the number of vimentin-positive cells/the number of DAPIstained nuclei. The purity of the isolated epithelial cells = the number of keratin-positive cells/the number of DAPI-stained nuclei.

Cell Culture
Fresh human ectopic and eutopic endometrial tissues were processed for cell isolation. The isolated endometrial stromal cells were cultured in DMEM/F12 with 10% FBS. The isolated endometrial epithelial cells were cultured in DKSFM. Epithelial cells were passaged only once, while stromal cells were used between passages 2 and 6. The endometrial cancer cell lines ECC-1 and Ishikawa were purchased from the American Type Culture Collection (ATCC, VA, USA). ECC-1 cells were grown in RPMI 1640 medium (Corning, Tewksbury, MA, USA) supplemented with 5% FBS. Ishikawa cells were grown in DMEM (Corning) supplemented with 10% FBS. The cells were maintained in a humidified incubator with an atmosphere comprising 5% CO 2 at 37°C. All media were supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin.
Telomeric Repeat Amplification Protocol (TRAP) Assay Telomerase activity was measured using the methods described by Wege et al. [23] and Herbert [24] with some modifications. Briefly, cell pellets were resuspended in CHAPS lysis buffer (Sigma-Aldrich, Dorset, UK) and incubated for 30 min on ice. After centrifugation at 16,000 g for 20 min at 4°C, the supernatant was collected and quantified using a BCA protein assay kit (Thermo Scientific, Loughborough, UK) according to the manufacturer's protocols. Then, the equivalent samples were placed in a plate and mixed with a master mix containing ×1 SYBR Green Master Mix (Thermo Scientific), TS primer (5′-AATCCGTCG AGCAGAGTT-3′) and ACX primer (5′-GCGCGGCTTACCCTT ACCCTTACCCTAACC-3′). The plate was incubated at 30°C for 30 min in the dark to extend the substrate by telomerase, and then PCR was carried out at 95°C for 10 min, followed by a 40-cycle amplification (95°C for 20 s, 50°C for 30 s, and 72°C for 90 s).

Cell Viability Assay
The endometrial stromal cells were plated and grown in 96-well plates at a concentration of 4,000 cells/well for 24 h. The cells were subsequently treated with varying doses of BIBR1532 for 72 h. MTT (5 mg/mL) was added to the 96-well plates at 5 µL/ well, followed by an additional hour of incubation. The MTT reaction was terminated by adding 100 µL of DMSO before the absorbance was read at 490 nm. The effect of BIBR1532 was calculated as a percentage of control cell growth obtained from DMSO-treated cells grown in the same 96-well plates.

Colony Formation Assay
The cells were seeded in 6-well plates (500 cells/dish) in their standard growth media. The cells were treated with BIBR1532 for 24 h after adherence overnight. Then, the cells were cultured for 12 days with media changes every third or fourth day. The plates were stained with 0.5% crystal violet, and colonies were counted under a microscope.

Annexin V Assay
Apoptosis was detected with a FITC Annexin V detection kit (BD Bioscience, San Jose, CA, USA) according to the manufacturer's protocols. Briefly, 1.5 × 10 5 cells/well were seeded into 6well plates, incubated overnight and then treated with BIBR1532 at different doses for 24 h. The cells were then collected, washed with PBS and resuspended in 100 µL binding buffer. Subsequently, 5 µL of Annexin V-FITC and 5 µL of propidium iodide were added to the binding buffer. After 15 min in the dark, the samples were analyzed by flow cytometry.

Cell Cycle Assay
The endometrial cells were plated in DMEM/F12 medium containing 10% FBS at a concentration of 1.5 × 10 5 cells/well in a 6-well plate for 24 h. The cells were subsequently treated with BIBR1532 at varying concentrations for 48 h. Then, the cells were collected, washed with PBS, fixed in a 70% ethanol solution and stored at 4°C overnight. The cell cycle assay was performed using BD cell cycle reagent (CycleTest ™ Plus DNA reagent kit, Becton Dickinson, Belgium) according to the manufacturer's protocol. Then, the samples were measured and analyzed by flow cytometry.

Wound Healing Assay
The cells were plated in 6-well plates at 3.5 × 10 5 cells/well for 24 h, and then a sterile 200-µL pipette tip was used to scratch a straight line across the plate in one direction. The cells were washed with fresh media to remove the detached cells and then treated with BIBR1532 at varying concentrations. The images were acquired after 0, 12 and 24 h of treatment. ImageJ was used to measure the area without cells. The migration rates at 12 and 24 h were calculated as follows: at 12 h=(the area at 12 hthe area at 0 h)/the area at 0 h; at 24 h=(the area at 24 hthe area at 0 h)/the area at 0 h.

Transwell Invasion Assay
Transwell assays were performed using 8-µm transwell chambers (Corning, Tewksbury, MA, USA) coated with Matrigel (BD Bioscience). The upper chamber with stromal cells (5 × 10 4 /well) and a lower chamber containing DMEM/F12 supplemented with 10% FBS were prepared. After 24 h, the cells were fixed and stained. Finally, invading cells were measured using an inverted microscope (Olympus Corporation, Tokyo, Japan). The relative cell invasion level was calculated as a percentage of untreated control cells.

Western Blot Analysis
The cells were plated at 2.0 × 10 5 cells/well in 6-well plates in their corresponding media and then treated with BIBR1532. Cell lysates were prepared in RIPA buffer. Equal amounts of protein were separated by gel electrophoresis and transferred onto PVDF membranes. The membrane was blocked for 1 h and then incubated with a 1:1,000 dilution of primary antibody overnight at 4°C. The membrane was then washed and incubated with the appropriate IRDyeTM 800-conjugated secondary antibody (1: 10,000) for 1 h after washing. Signals were detected using the Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE, USA) and analyzed with Odyssey software. After developing, the membrane was stripped and reprobed using antibodies against β-actin or GAPDH to confirm equal loading. The intensity of each band was measured and normalized to that of β-actin or GAPDH as an internal control. The antibodies used in the study were as follows

Enrichment Analysis
The gene expression profiles of GSE120103 and GSE5108 were obtained from the Gene Expression Omnibus (GEO) database.
GSE120103, based on the GPL6480 platform, included endometria from women without and with endometriosis [25]. The GSE5108 dataset was produced using the GPL2895 platform, which contains a gene expression microarray of eutopic and ectopic endometria [26].
GEO2R, an interactive tool used for comparing two groups of samples, was used to identify the DEGs in the GSE120103 and GSE5108 datasets with the limma package. The genes with a fold change >1 and an adj.p value <0.05 were selected for enrichment analysis using Metascape. Then, the gene set database of GSEA named "GOBP_POSITIVE_REGULATION_OF_MAPK_CAS-CADE" was selected, and GSEA was used to detect whether mitogen-activated protein kinase (MAPK) cascade signaling was enriched during endometriosis progression.

Statistical Analysis
The analysis was performed using GraphPad Prism 8. For the data derived from telomeric repeat amplification protocol, MTT, migration, and Western blot, control data were set at 1, and treatment groups were presented as the fold change of the control data. An unpaired Student's t test was used to compare the two groups. p values <0.05 were considered statistically significant.

Morphology of Isolated Human Endometrial Cells Was Altered by BIBR1532
Vimentin and cytokeratin are specific markers of human endometrial stromal cells and epithelial cells, respectively. As shown in Figure 1a, b, human endometrial stromal cells, which displayed long spindles and typical fibroblast-like shapes, positively expressed the mesenchymal-specific marker vimentin, while human endometrial epithelial cells grew in clumps with cytokeratin stained positively. The purity of the isolated human endometrial stromal cells and epithelial cells was >95% and >90%, respectively.
The morphology of the endometrial stromal cells and the epithelial cells derived from the patients with endometriosis was altered after treatment with BIBR1532 for 24 h. Stromal cells displayed decreased cell antennae, while epithelial cells shrank to a round shape (Fig. 1c, d), indicating that endometrial cells from the patients with endometriosis may be affected by BIBR1532, and its effect was further investigated.
The inhibitory effect of BIBR1532 on endometrial stromal cells was assessed by MTT assay (online suppl. Table II). HEuESCs and HEcESCs were exposed to varying doses of BIBR1532 for 72 h. The results showed that eutopic and ectopic endometrial stromal cells responded similarly to the agent and displayed obviously decreased viability with increasing concentrations of BIBR1532 (Fig. 2c, d).
Next, colony formation assays were performed to investigate the long-term effect of BIBR1532 on the proliferation of endometrial cells. Both colony number and colony size were decreased with BIBR1532 treatment in a dose-dependent manner (Fig. 2e, f).

BIBR1532 Affected Apoptosis and Cell Cycle Progression of Endometrial Cells
To determine the effect of BIBR1532 on apoptosis of endometrial cells, flow cytometry was used to test the expression of Annexin V on cell membranes. The results showed that BIBR1532 affected apoptosis of endometrial eutopic and ectopic stromal cells in a dose-dependent manner (online suppl. Table III). Treatment with BIBR1532 at different concentrations for 24 h resulted in a significant induction of apoptosis relative to the control in eutopic and ectopic endometrial stromal cells derived from endometriosis patients, as determined by the Annexin V assay (Fig. 3a, b). Western blotting results showed that BIBR1532 decreased the expression of the antiapoptotic genes Bcl-xl and Mcl-1 in both cell lines after 24 h of treatment, further confirming this finding (Fig. 3c, d).
Additionally, we also examined the effect of BIBR1532 on cell cycle progression using flow cytometry. As illustrated in Fig. 3e, f, exposure of eutopic endometrial stromal cells to 200 µm BIBR1532 for 48 h resulted in a decrease in G1 phase and an increase in S phase; however, no significant difference in cell cycle distribution was found in ectopic endometrial stromal cells treated with varying concentrations of BIBR1532 for 48 h.

BIBR1532 Suppressed the Migration and Invasion of Endometrial Cells
The wound healing assay demonstrated that the migratory capacity of eutopic stromal cells was stronger   Fig. 4. BIBR1532 inhibited endometrial cell migration and invasion. a-e Representative images reflecting the migratory capacity of HEuESCs (a) and HEcESCs (b) are presented, and the difference between eutopic and ectopic stromal cells (c) as well as the inhibitory effect of the agent is measured (d, e). f, g BIBR1532 also exhibited a similar reduction in cell invasion with BIBR1532 treatment in a concentration-dependent manner as detected by the transwell assay (n = 6) (f), and the corresponding histograms are presented (g). HEuESCs, human eutopic endometrial stromal cells; HEcESCs, human ectopic endometrial stromal cells. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

BIBR1532 Inhibits Endometriosis via Telomerase Inhibition and MAPK Signaling
Gynecol Obstet Invest 2023;88:226-239 DOI: 10.1159/000530460 than that of ectopic stromal cells (Fig. 4a-c) and that compared with untreated cells, treatment with BIBR1532 at varying concentrations for 12 h and 24 h decreased the motility of endometrial cells in both cell lines (Fig. 4d, e). In addition, eutopic and ectopic endometrial stromal cells exhibited a similar reduction in cell invasion with BIBR1532 treatment in a concentration-dependent manner as detected by transwell assay (Fig. 4f, g).

MAPK Signaling Was Associated with Endometriosis Progression and Was Restrained by BIBR1532
Previous studies have noted that the MAPK signaling pathway plays important roles in cellular events associated with endometriosis formation and progression, such as cell proliferation, cell differentiation, apoptosis, migration, angiogenesis, and the inflammatory response [27][28][29]. In this study, enrichment analysis using Metascape software showed that both the DEGs obtained from GSE120103 and GSE5108 were enriched in the MAPK cascade (Fig. 5a, b). The enrichment analysis of the GSEA gene set database named "GOBP_POSITIVE_REGULA-TION_OF_MAPK_CASCADE" also showed that compared with endometrial samples from women without endometriosis, endometrial samples from patients with endometriosis appeared to be positively correlated with the MAPK cascade (p = 0.33), although the difference was not significant (Fig. 6a); the DEGs between the ectopic and eutopic endometrial samples were markedly enriched in the MAPK cascade (p = 0.036) (Fig. 6b).
MAPK signaling mainly includes three signal cascades: extracellular signal-regulated kinase (ERK), p38, and JNK [30]. To evaluate the effect of BIBR1532 on MAPK signaling, we treated HEuESCs and HEcESCs with various doses of BIBR1532 and measured the effect of BIBR1532 on ERK1/2, p38, and JNK. The results showed that BIBR1532 induced a significant decrease in the protein levels of phospho-p44/42, phospho-p38, and phospho-JNK (Fig. 6c, d), different from those observed for the endometrial cell lines, among which both ECC-1 and Ishikawa cells displayed increases in the protein levels of phospho-p44/42, phospho-p38, and phospho-JNK (online suppl. Fig. S1).

Discussion
Telomerase is a ribonucleoprotein complex consisting of three main components: TERT, the core catalytic subunit of telomerase; telomerase RNA component, a template for telomere synthesis; and several associated proteins, such as dyskerin [31,32]. It is well known that the main function of telomerase is to add DNA sequence repeats (TTAGGG) to the 3′ end of chromosomes to maintain the length of telomeres and thereby prevent cell cycle arrest and cell death caused by telomere shortening [31,32]. Kim et al. [10] and Hapangama et al. [11] found that increased TA and high TL are observed in the eutopic endometrium of women with endometriosis. Valentijn et al. [12] and Alnafakh et al. [13] found that human endometrial cell proliferation was associated with high TA and increased telomerase component expression. The establishment of endometriosis lesions in a naked monkey model was reported to be associated with the induction of high telomerase expression [33]. DEGs associated with telomeres and telomerase between normal endometrial samples and endometrial samples from patients with endometriosis have also been identified [13]. These findings indicated the role of telomerase in endometriosis and the potential application value of telomerase inhibition in endometriosis therapy; however, related studies are lacking. Therefore, in this study, the telomerase inhibitor BIBR1532 was used to preliminarily explore the effect of telomerase inhibition on endometriotic cells.
Endometriosis is a complex disease arising from the interactions among multiple factors, such as immune cells, adhesion molecules, extracellular matrix metalloproteinases, and proinflammatory cytokines [1]. The dysregulation of cell proliferation and apoptosis induced by these factors promotes the ectopic survival of the endometrial cells [34,35]. The cytostatic effect of BIBR1532 has been reported previously in multiple papers [15,21,36,37]. The results of this study also showed that treatment with BIBR1532 inhibits the growth of eutopic and ectopic endometrial stromal cells derived from patients with endometriosis in a dose-dependent manner, accompanied by the induction of apoptosis. The inhibition rate of cell proliferation and telomerase activity induced by BIBR1532 was similar, suggesting that both eutopic and ectopic endometrial stromal cells are telomerase addicted in their proliferative abilities. The results of the colony formation assay, which displayed a decrease in colony number and colony size in a dose-dependent manner, also indicated this effect. Previous studies also reported the involvement of cell cycle arrest in the cytostatic effect of BIBR1532 [15,21,36,37], which was also found in the current study. This is consistent with the series of responses to telomere attrition, which triggers the DNA damage response pathway and subsequently leads to cell cycle arrest and apoptosis [38][39][40]. These findings suggest that the cytostatic effect of BIBR1532 is likely triggered by a series of responses through telomere shortening because of the failure of telomere elongation by telomerase. However, the triggered cell cycle change is varied. BIBR1532 can induce the S-phase accumulation of Epstein-Barr virusimmortalized and transformed B cells [18] but blocks the cell cycle in the G0/G1 phase in chronic myeloid leukemia cells and glioma cells [41]. In this study, BIBR1532 treatment induced different cell cycle changes in cells originating from different endometriosis patients, and even if derived from the same patient, eutopic and ectopic endometrial cells responded differently to the agent, illustrating that different gene expressions in the cells may contribute to different responses to the agent. Previous studies also reported that different primary cells, even those derived from patients with the same type of disease, showed different sensitivities to BIBR1532 [42]. Likewise, in the colony formation assays, the colony size was different with the same concentration of the agent. So, identification of the genes associated with the effects of drug action may be beneficial for its application and can be further investigated in the future. According to retrograde menstruation theory, to develop endometriosis, live endometrial cells migrate through the fallopian tubes and are implanted on the surface of the pelvic cavity [6,43], indicating that the ability of endometrial cell migration and invasion is also important for the establishment of ectopic lesions. The ability of migration and invasion, which is being studied in endometriosis by many studies, is also the biological behavior of endometriotic cells, which is similar to malignant cells [44,45]. Therefore, in this study, in addition to the effect of BIBR1532 on cell proliferation, its effects on cell migration and invasion were also tested. We found that BIBR1532 inhibited the migration and invasion of eutopic and ectopic endometrial stromal cells, meaning that telomerase inhibition can not only inhibit the growth of ectopic lesions but also prevent the occurrence of endometriomas.
To explore the pathways of the effect of telomere shortening caused by telomerase inhibition on cell viability, migration, and invasion, we enriched the DEGs and found that the MAPK cascade was involved in the aberrations in eutopic endometrial samples derived from patients with endometriosis, which can be further confirmed by GSEA results. Adewuyi and Sapkota [46] also found MAPK signaling enrichment in endometriosis using genome-wide association study data, and abnormal activation of MAPK signaling in eutopic stromal cells of patients with endometriosis has been observed [47]. MAPK is a group of serine/threonine kinases that can be activated by extracellular stimuli, such as cytokines, hormones, and cell stress [48]. A pan-cancer analysis has uncovered the correlation between amplified MAPK signaling and high telomerase activity [49]. It has also been reported that MAPK expression is associated with telomerase promoter mutations, and mutations can determine therapeutic responses to MEK inhibitors [50][51][52]. MAPK is a complicated intracellular signal transduction pathway [30]. MAPK participates in the maintenance of telomeres and can influence telomerase activity [53,54]. Bi et al. [55] found that irisin can increase telomerase activity via inhibition of JNK phosphorylation. Lanna et al. [56] found that IFN-α activates p38 MAPK signaling, which inhibits telomerase activity. However, TERT also transmits signaling through MAPK, and a positive feedback loop may develop, such as PI3K/ AKT/mTOR and NF-kB with TERT [54]. In this study, telomerase inhibition by BIBR1532 inhibited the phosphorylation of ERK, p38, and JNK in eutopic and ectopic endometrial stromal cells, while for the epithelial cells ECC-1 and Ishikawa, the responses were reversed, indicating that different signaling pathways are involved in the drug action.
The observed effects of the telomerase inhibitor on eutopic and ectopic endometrial stromal cells derived from patients with endometriosis suggested the potential clinical value of telomerase inhibition in endometriosis therapy. However, selecting an appropriate drug delivery method was necessary for its clinical application, and we will further investigate it.