Invasion Of Endometriotic Cells Research Articles

MCP-1 exerts the inflammatory response via ILK activation during endometriosis pathogenesis

AimsMCP-1 has been shown to be elevated in endometriosis. ILK functions in several cellular events and interacts with MCP-1-signaling. In the current study, we evaluated the role of MCP-1-ILK signaling in human endometriotic cell's (Hs832(C).TCs) potential for colonization, invasion, adhesion, etc. and differentiation of macrophage along with inflammation in an endometriosis mouse model. Materials and methodsA mouse model of endometriosis with elevated levels of MCP-1 was developed by injecting MCP-1. We examined the migration, adhesion, colonization and invasion of Hs832(C).TCs in response to MCP-1-ILK signaling. We also examined the differentiation of THP-1 cells to macrophage in response to MCP-1-ILK signaling. Key findingsWe observed that MCP-1 increased Ser246 phosphorylation of ILK in Hs832(C).TCs and enhanced the migration, adhesion, colonization, and invasion of Hs832(C).TCs. In the mouse model of endometriosis, we found elevated chemokines (CCL-11, CCL-22 and CXCL13) levels. An increased level of MCP-1 mediated ILK activation, leading to increased inflammatory reaction and infiltration of residential and circulatory macrophages, and monocyte differentiation, but suppressed the anti-inflammatory reaction. The inhibitor (CPD22) of ILK reversed the MCP-1-mediated action by restoring Hs832(C).TCs and THP-1 phenotype. ILK inhibition in a mouse model of endometriosis reduced the effects of MCP-1 mediated pro-inflammatory cytokines, but increased anti-inflammatory response along with T-regulatory and T-helper cell restoration. SignificanceTargeting ILK restores MCP-1 milieu in the peritoneal cavity and endometrial tissues, reduces the inflammatory response, improves the T-regulatory and T-helper cells in the endometriosis mouse model and decreases the migration, adhesion, colonization and invasion of endometriotic cells.

Identification of diagnostic markers related to inflammatory response and cellular senescence in endometriosis using machine learning and in vitro experiment.

To understand the association between chronic inflammation, cellular senescence, and immunological infiltration in endometriosis. Datasets from GEO comprising 108 endometriosis and 97 healthy human samples and the human endometrial stromal cell. Differentially expressed genes were identified using Limma and WGCNA. Inflammatory response-related subtypes were constructed using consensus clustering analysis. The CIBERSORT algorithm and correlation analyses assessed immune cell infiltration. LASSO, SVM-RFE, and RF identified diagnostic genes. Functional enrichment analysis and multifactor regulatory networks established functional effects. Nomograms, internal and external validations, and in vitro experiments validated the diagnostic genes. Inflammatory response subtypes were highly correlated with the immune activities of B and NK cells. Sixteen genes were associated with inflammatory response and cellular senescence and six diagnostic genes (NLK, RAD51, TIMELESS, TBX3, MET, and BTG3) were identified. The six diagnostic gene models had an area under the curve of 0.828 and their expression was significantly downregulated in endometriosis samples. Low expression of NLK and BTG3 promoted the proliferation, migration, and invasion of endometriotic cells. Inflammatory response subtypes were successfully constructed for endometriosis. Six diagnostic genes related to inflammatory response and cellular senescence were identified and validated. Our study provides novel insights for inflammatory response in endometriosis and markers for endometriosis diagnosis and treatment.

The significance of immune microenvironment in patients with endometriosis.

Endometriosis represents an estrogen-dependent disease of the female reproductive system and intra- and extraperitoneal regions, with chronic feature. Currently, immune cells, such as macrophages and lymphocytes, are considered to play a pivotal role in angiogenesis and invasion of endometriotic cells through matrix remodeling. Additionally, various studies have revealed the role of E-cadherin, β-catenin, along with steroid hormone receptors in endometriosis development. In this context, our study aimed to analyze the relationship between the cellular immune profile and E-cadherin, β-catenin, estrogen receptor alpha (ERα), and progesterone receptor (PR) immunoexpression in endometriosis tissues, along with an analysis of the possible association between serological parameters and immunohistochemical (IHC) markers. The study included 53 patients diagnosed with ovarian or cutaneous abdominal wall endometriosis, which have been investigated by routine histology, immunohistochemistry, and serum analysis. The IHC exam showed an increased density of cluster of differentiation (CD)4+ T-cells, CD8+ T-cells, and CD68+ macrophages, along with variable increased expressions of E-cadherin, β-catenin, ERα, and PR. Statistical analysis revealed an intense positive correlation between CD68 and PR expression (p<0.05), without any other statistically significant correlations between IHC markers or between IHC and serological markers. Our study supports that endometriosis is an immune-dependent disease characterized by an abnormal morphological profile of T-cells and macrophages in endometriotic implants. Our study provides additional data useful in the understanding the immune milieu of endometriosis in the context of its complex pathogenic molecular mechanism. Further research is needed to develop new immunological therapeutic approaches, like immune checkpoint inhibitors administration or T-cell-targeted immunotherapy in these patients.

Upregulation of SPI1 in Ectopic Endometrium Contributes to an Invasive Phenotype

Endometriosis is one of the most common gynecological diseases associated with chronic pelvic pain, infertility, and cancer. However, its molecular pathogenesis remains unclear. This study aimed to identify key genes involved in the pathogenesis of endometriosis. Bioinformatic analyses were perfomed to identify key differentially expressed genes (DEGs), transcription factors (TFs), and functionally enriched pathways. Effect of SPI1 on migration, invasion, expression of ADH1B, MYH11, and PLN were analyzed in human endometriotic cells. By screening three transcriptome datasets from the GEO for overlapping DEGs between eutopic and ectopic endometria in patients with endometriosis, we found that the expression of ADH1B, MYH11, and PLN was markedly upregulated in the ectopic endometrium. Knockdown of ADH1B, MYH11, and PLN significantly inhibited the migration and invasion of human endometriotic 12Z cells. Additionally, gene set enrichment analysis revealed that epithelial-mesenchymal transition gene signature was positively correlated with ADH1B, MYH11, and PLN expression. Notably, the TF SPI1 was found to regulate the expression of these three genes in the endometriotic tissues and 12TZ cells. Moreover, SPI1 expression was associated with the invasion of endometriotic cells and was increased in the ectopic endometrium of patients with endometriosis. These data suggest that SPI1 plays a key role in the progression of endometriosis by regulating ADH1B, MYH11, and PLN expression and may therefore serve as a potential prognostic and therapeutic factor for endometriosis.

The ellagic acid metabolites urolithin A and B differentially affect growth, adhesion, motility, and invasion of endometriotic cells in vitro.

The ellagic acid metabolites urolithin A and B differentially affect growth, adhesion, motility, and invasion of endometriotic cells in vitro.

Propofol inhibits proliferation and invasion of endometriotic cells by miR-9-5p/TGFBI axis

Endometriosis (EM) is an estrogen-dependent, frequent benign gynecological disease, which reduces the quality of life in EM patients. Propofol regulates the apoptosis of EM. miR-9-5p is decreased in early secretory endometrium, TGFBI is increased in EM, however, their functions in EM remain unclear. This study aimed to investigate the mechanisms underlying the effects of propofol on the proliferation and invasion of EM by regulating miR-9-5p/TGFBI. The proliferation and invasion of ectopic endometrial stromal cells (ESCs) were determined by MTT assay and Transwell assay, respectively. Real-time PCR was applied for the evaluation of miR-9-5p and transforming growth factor-β (TGF-β)-induced gene (TGFBI) mRNA level in ESCs. Western blot was used to examine the protein level of TGFBI protein (TGFBIp) in ESCs. The relation between miR-9-5p and TGFBI was predicted by TargetScan 7.1 and verified by dual luciferase reporter assay. Propofol reduced cell proliferation and invasion, while induced miR-9-5p level and reduced TGFBI/TGFBIp level in ectopic ESCs. Additionally, in ectopic ESCs from patients with EM, miR-9-5p was lower, while TGFBI and TGFBIp were higher than those from eutopic endometrium and normal endometrium from patients with hysteromyoma. Moreover, propofol reduced proliferation and invasion in ectopic ESCs, which was realized by the regulation of miR-9-5p/TGFBI.

Upregulation of Fibroblast Growth Factors Caused by Heart and Neural Crest Derivatives Expressed 2 Suppression in Endometriotic Cells: A Possible Therapeutic Target in Endometriosis.

Several features exist that distinguish endometriotic cells from eutopic endometrial cells. Progesterone resistance is one of the main distinguishing features, although how progesterone resistance affects the phenotype of endometriotic cells is not fully elucidated. Heart and neural crest derivatives expressed 2 (HAND2) is a transcriptional factor that plays an important role in maintaining endometrial function in a progesterone-dependent manner. Therefore, we explored whether progesterone-dependent HAND2 is implicated in the progression of endometriosis. HAND2 was less expressed by endometriotic tissues compared to endometrial tissues. Suppression of HAND2 expression induced fibroblast growth factor 1 (FGF1), FGF2, and FGF9 in endometriotic stromal cells and consequently enhanced migration and invasion capacity. AZD4547, a FGF receptor inhibitor, diminished the migration and invasion of endometriotic cells in vitro. In the murine model of endometriosis, AZD4547 showed suppressive effects on the development of endometriotic lesions at a relatively low concentration. In conclusion, we demonstrated that FGF1, FGF2, and FGF9 are downstream effectors of HAND2 in endometriotic cells. Since HAND2-dependent FGFs play roles in enhancing invasive capacity of endometriotic cells, our results suggest that FGF receptor inhibitors, such as AZD4547, can be promising therapeutic targets for endometriosis.

Effects of histone methyltransferase inhibition in endometriosis.

Although the histone methyltransferase EZH2 and its product H3K27me3 are well studied in cancer, little is known about their role and potential as therapeutic targets in endometriosis. We have previously reported that endometriotic lesions are characterized by global enrichment of H3K27me3. Therefore, we aimed to (1) characterize the expression levels of EZH2 in endometriotic tissues; (2) assess H3K27me3 enrichment in candidate genes promoter regions; and (3) determine if pharmacological inhibition of EZH2 impacts migration, proliferation, and invasion of endometriotic cells. Immunohistochemistry of an endometriosis-focused tissue microarray was used to assess the EZH2 protein levels in tissues. Chromatin immunoprecipitation-qPCR was conducted to assess enrichment of H3K27me3 in candidate gene promoter regions in tissues. Immunofluorescence was performed to assess the effect of an EZH2-specific pharmacological inhibitor on H3K27me3 global enrichment in cell lines. To measure effects of the inhibitor in migration, proliferation, and invasion in vitro we used Scratch, BrdU, and Matrigel assays, respectively. Endometriotic lesions had significantly higher EZH2α nuclear immunostaining levels compared to eutopic endometrium from patients (glands, stroma) and controls (glands). H3K27me3 was enriched within promoter regions of candidate genes in some but not all of the endometriotic lesions. Inhibition of EZH2 reduced H3K27me3 levels in the endometriotic cells specifically, and also reduced migration, proliferation but not invasion of endometriotic epithelial cells (12Z). These findings support future preclinical studies to determine in vivo efficacy of EZH2 inhibitors as promising nonhormonal treatments for endometriosis, still an incurable gynecological disease.

Pharmacological blockage of the CXCR4-CXCL12 axis in endometriosis leads to contrasting effects in proliferation, migration, and invasion.

High levels of inflammatory factors including chemokines have been reported in peritoneal fluid and blood of women with endometriosis. CXCL12 mediates its action by interaction with its specific receptor, CXCR4, reported to be elevated in human endometriosis lesions and in the rat model of endometriosis. Activation of the CXCR4-CXCL12 axis increases cell proliferation, migration, and invasion of cancer cells. To obtain insights into the CXCR4 expression profile in lesions and endometrium, as well as functionality of the CXCR4-CXCL12 axis in endometriosis, we analyzed the expression of CXCR4 in tissues on a human tissue array and studied CXCL12-mediated activation of proliferation, invasion, and migration in vitro. We observed differences in levels of nuclear CXCR4 expression among lesion types, being higher in ovarian lesions. Endometriotic cell lines (12Z) showed higher levels of CXCR4, proliferative and migratory potential, and AKT phosphorylation/kinase activity compared to untreated control cells (endometrial epithelial cells). CXCL12 and endometriotic stromal cell-enriched media increased proliferation of non-endometriotic epithelial cells. CXCL12 caused a significant increase in 12Z cell invasion but had no effect on migration; AMD3100, a CXCR4-specific inhibitor, significantly increased invasion of 12Z cells but decreased their migration. However, treatment with CXCL12 plus AMD3100 significantly decreased invasion and migration of 12Z cells. In conclusion, the CXCR4-CXCL12 axis is functional in endometriosis cells, but the expression of CXCR4 varies among lesions. CXCL12 promoted proliferation, migration, and invasion of endometriotic cells, while inducing AKT phosphorylation and activity, but pharmacologically blocking this axis in the absence of the ligand induced their invasiveness.

Overexpression of microRNA-542-3p attenuates the differentiating capacity of endometriotic stromal cells.

AimEndometriosis is defined as the presence of endometrial glandular and stromal cells outside of the uterine cavity. A previous study reported that microRNA (miR)‐542‐3p plays a critical role in eutopic endometrial decidualization. This study aims to clarify the potential role of miR‐542‐3p and the target gene, IGFBP‐1 (insulin‐like growth factor‐binding protein 1), in the impairment of the decidualizing capacity of human ectopic endometrial stromal cells (HEcESCs).MethodsIn vitro analysis of primary undifferentiated and decidualizing human eutopic endometrial stromal cells (HEuESCs) and HEcESCs was conducted. The primary HEuESCs or HEcESCs were expanded in culture and decidualized with 8‐bromo‐cyclic adenosine monophosphate (8‐bromo‐cAMP) and medroxyprogesterone acetate (MPA).ResultsThe morphological and biological differentiating capacities of the HEcESCs were markedly impaired. In contrast to the HEuESCs, the HEcESCs that were treated with the decidual stimulus retained the mesenchymal phenotype and capacity for migration. The down‐regulation of miR‐542‐3p in the HEcESCs treatment with 8‐bromo‐cAMP and MPA was much weaker than that of the HEuESCs. High expression of miR‐542‐3p led to a significant decrease in the expression of IGFBP1 in the HEcESCs.ConclusionImpairment of the differentiating capacity by the overexpression of miR‐542‐3p could influence the capacity for migration and invasion of endometriotic cells in an ectopic environment.

Leptin promotes human endometriotic cell migration and invasion by up-regulating MMP-2 through the JAK2/STAT3 signaling pathway.

Despite evidence that leptin may play a role in the pathogenesis of endometriosis, the specific function of leptin in the migration and invasion of endometriotic cells is not well characterized. In this study, we investigated the effect of leptin on the migration, invasion and matrix metalloproteinase (MMP) expression levels of human endometriotic cells. We found that leptin stimulated the migration and invasion of endometriotic cells (11Z, 12Z and 22B) in a dose-dependent manner. Leptin receptor (ObR) siRNA significantly inhibited the migration and invasion induced by leptin in 11Z and 12Z cells. Leptin-induced migration and invasion were significantly attenuated by pretreatment with SB-3CT, a specific gelatinase (MMP-2 and MMP-9) inhibitor. In addition, leptin-induced increases in the mRNA and protein expression and enzyme activity of MMP-2 in 11Z and 12Z cells. Selectively inhibiting MMP-2 using siRNA and an inhibitor (GM6003), impaired the ability of leptin to stimulate the migration and invasion of endometriotic cells, suggesting that MMP-2 plays an essential role in leptin-induced migration and invasion. Janus Kinase 2/Signal Transducer and Activator of Transcription 3 (JAK2/STAT3) inhibitor (AG490) significantly inhibited the migration, invasion and MMP-2 expression induced by leptin in endometriotic cells. Furthermore, the Extracellular signal-Regulated Kinase inhibitor PD98059 neutralized the migration and invasion promoting effects of leptin. Taken together, these results suggest that leptin may contribute to the migration and invasion abilities of endometriotic cells via the up-regulation of MMP-2 through an ObR-dependent JAK2/STAT3 signaling pathway.

CXCR4‐CXCL12 axis in proliferation, migration, and invasion of endometriosis (LB850)

Endometriosis a benign gynaecological disease, associated with chronic inflammation and increased levels of chemokines. Lesions are composed of endometrial stroma and glands localized outside the uterus. In a rat model of endometriosis, we showed high CXCR4 mRNA levels in endometriotic lesions and in high protein expression levels in human endometriosis tissues compared to control endometrium . The CXCR4‐CXCL12 axis plays roles in proliferation, migration, and cell invasion. Endometriotic epithelial cells expressed CXCR4. We hypothesized that CXCL12 may induce proliferation, migration, and invasion of endometriotic cells (12Z) compared to a non‐invasive breast cancer cells, expressing endometrial markers (EEC), and the induction may be blocked by the CXCR4 inhibitor, AMD3100. Cell proliferation in response to CXCL12 was analyzed by BrdU assay. We also studied cell invasion and migration in response to CXCL12 and AMD3100 by invasion and migration chambers. Rat model of endometriosis was used to identify the effect of AMD3100 in lesions regression. CXCL12 increased proliferation and migration of EEC, and invasion in 12Z cells, compared to media (p&lt;0.05). AMD3100 reduced migration in EEC; reduced migration and increased invasion in 12Z cells (p&lt;0.05). These data suggest that the CXCR4‐CXCL12 axis can be activated in endometrial cells to promote cell proliferation, migration, and invasion, and application of cancer treatments to endometriosis disease requires additional studies. These biological functions have been previously shown to be dysregulated in endometriosis, by still undefined mechanisms, leading to survival and growth of ectopic endometrium.Grant Funding Source: Supported by F31HD072594, RCMI G12‐MD007579, R25GM082406, R25GM096955, R01‐HD050509

Targeting of Syndecan-1 by microRNA miR-10b modulates invasiveness of endometriotic cells via dysregulation of IL-6 secretion and MAPK signaling

Targeting of Syndecan-1 by microRNA miR-10b modulates invasiveness of endometriotic cells via dysregulation of IL-6 secretion and MAPK signaling

Selective inhibition of prostaglandin E 2 signaling inhibits migration and invasion of human endometriotic epithelial and stromal cells through multiple mechanisms

OBJECTIVE: Prostaglandin E2 (PGE2) plays central role in the pathogenesis of endometriosis. We have shown that selective inhibition of COX-2 prevents survival, migration, and invasion (Endocrinology, 2008), and inhibition of EP2 and EP4 induces apoptosis (Molecular Endocrinology, 2009) of human endometriotic epithelial and stromal cells. In this study, we determined molecular association between PGE2 signaling and migration and invasion potential of human endometriotic cells. DESIGN: Human endometriotic and endometrial tissues from proliferative phase of the menstrual cycle and human immortalized endometriotic epithelial cells 12Z and stromal cells 22B. MATERIALS AND METHODS: Spatial expression of PGE2 receptors EP1, EP2, EP3 and EP4 were studied in human ectopic (n=12) and eutopic endometria (n=12) using immunohistochemistry. PGE2 signaling was inhibited using pharmacological and genomic approaches in endometriotic epithelial cells 12Z and stromal cells 22B, and their effects on migration and invasion of endometriotic cells and the underlined molecular mechanisms were determined (n=3). RESULTS: Our data indicated that EP2 and EP4 were highly (P<0.05) expressed in glandular epithelium and stoma of ectopic compared to eutopic endometria. EP1 was expressed at very low level in both ectopic and eutopic endometria. EP3 was undetectable in both endometria. Selective inhibition of EP2 and EP4 receptors inhibited (P<0.05) migration and invasion of endometriotic epithelial cells 12Z and stromal cells 22B in a cell-specific manner. This effect was mediated through inhibition of metalloproteinases MMP2 and MMP9 activities via suppression of ERK1/2, AKT, NFkB and b-catenin pathways. CONCLUSIONS: Our results suggest that selective inhibition of EP2 and EP4 could be used as novel non-estrogen hormonal therapy for endometriosis, pending in vivo studies.

Cyclooxygenase-2 overexpression in ovarian endometriomas is associated with higher risk of recurrence

Cyclooxegenase-2 expression was evaluated by immunohistochemistry in endometrioma tissue samples taken from 109 patients, of whom 53 had recurrence by 30 months after surgery and 56 did not. Cyclooxegenase-2 overexpression, along with previous medical treatment of endometriosis and the presence of adhesion, is predictive of recurrence of ovarian endometrioma within 30 months after surgery, with a resultant sensitivity of 72.5% and a specificity of 72.4%, suggesting that there are intrinsic and identifiable biomarkers that confer recurrence risk.

Cyclooxygenase-2 Regulates Survival, Migration, and Invasion of Human Endometriotic Cells through Multiple Mechanisms

Endometriosis is a debilitating disease characterized by the presence of functional endometrial glandular epithelium and stroma outside the uterine cavity that affects up to 20% of women of child-bearing age. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme in the biosynthesis of prostaglandin E(2) (PGE(2)), is highly expressed in endometriotic tissues and results in increased concentrations of peritoneal PGE(2) in women. In this study, we determined the expression of COX-2 protein in ectopic and eutopic endometria in humans and the role of COX-2 in endometriotic cell survival, migration, and invasion in humans. Our results indicate that COX-2 protein is abundantly expressed in ectopic endometria compared with eutopic endometria. Comparatively, expression of COX-2 protein is higher in eutopic endometria from women with endometriosis compared with women without endometriosis. Inhibition of COX-2 decreases survival, migration, and invasion of endometriotic cells that are associated with decreased production of PGE(2). Cell growth inhibitory effects of COX-2 inhibition/silencing are mediated through nuclear poly (ADP-ribose) polymerase-mediated apoptosis. Cell motility and invasion inhibitory effects of COX-2 inhibition/silencing are mediated through matrix metalloproteinase-2 and -9 activities. Interestingly, effects of COX-2 inhibition is more profound in endometriotic epithelial than in stromal cells. Furthermore, inhibition of COX-2 affects invasion rather than migration of endometriotic epithelial and stromal cells. It is the first evidence showing that inhibition of COX-2 decreases endometriotic epithelial and stromal cell survival, migration, and invasion in humans. Our results support the emerging concept that COX-2/PGE(2) promotes the pathophysiology and pathogenesis of endometriosis in humans.

Study on the invasiveness of endometriotic cells in vitro

Objective: The most acceptable theory on the pathogenesis of endometriosis is reflux of menstrual flow invade other tissue and implant at abnormal sites. So, we design to compare the invasiveness between eutopic and ectopic endometrium in vitro.

Invasiveness of endometriotic cells in vitro.

The pathogenesis of endometriosis is not known. The currently favoured theory is that viable endometrial cells, shed from the endometrium into the pelvic cavity by retrograde menstruation, reattach and invade other tissues. We used a collagen gel invasion assay to assess invasive potential of endometriotic cells. The invasion indices of cells from peritoneal endometriotic lesions and a metastatic bladder carcinoma cell line (EJ28) were similar (2.2-15.6 vs 8.4-11.6) whereas cells from normal endometrium and non-metastatic carcinoma cells (RT112) were non-invasive (indices < 1). Invasiveness of endometriotic cells might contribute to the pathogenesis of endometriosis.