Therapeutic Target For Endometriosis Research Articles

Notch activity mediates oestrogen-induced stromal cell invasion in endometriosis.

Oestrogen has been reported to control the invasiveness of endometrial stromal cells in endometriosis. Notch signalling, a master regulator of cell invasion in tumours, is regulated by oestrogen in other diseases and hyperactivated in endometriotic stromal cells. Therefore, we hypothesized that an interaction between Notch signalling and oestrogen may exist in the regulation of endometrial stromal cell invasion, which is essential for the development of endometriosis. Western blot analysis of tissues showed that the expression levels of Notch components (JAG1 and NOTCH1) and Notch activity were markedly higher in ectopic endometria than in their eutopic and normal counterparts. Primary stromal cells obtained from normal endometria cultured with oestrogen presented significant increases in the expression of Notch components and Notch activity, the cytoplasmic and nuclear accumulation of NOTCH1 intracellular domain, the expression of matrix metallopeptidase 9 and vascular endothelial growth factor and cell invasiveness. Knockdown of NOTCH1 markedly alleviated oestrogen-induced matrix metallopeptidase 9 and vascular endothelial growth factor expression and cell invasion. ICI (an oestrogen receptor α antagonist) also blocked these oestrogenic effects. Oestrogen-responsive elements were found in the promoters of NOTCH1 and JAG1. A luciferase reporter analysis revealed that oestrogen regulated the expression of Notch components via oestrogen receptor alpha, which is bound to oestrogen-responsive elements in the JAG1 and NOTCH1 promoters. Collectively, our findings indicate that oestrogen engages in crosstalk with Notch signalling to regulate cell invasion in endometriosis via the activation of oestrogen receptor alpha and the enhancement of Notch activity. Notch signalling blockade may therefore be a novel therapeutic target for endometriosis.

Inhibition of IAP (inhibitor of apoptosis) proteins represses inflammatory status via nuclear factor-kappa B pathway in murine endometriosis lesions.

How is the role of inhibitor of apoptosis proteins (IAPs) in the development of murine endometriosis lesions? BALB/c female mice (n=36) were used for the murine endometriosis model. Endometriotic lesions were surgically induced in mice by transplanting mouse uterine tissue. After 4weeks of IAP antagonist (BV6) treatment, the expression of inflammatory factors in the implants was evaluated using real-time RT-PCR. Inflammatory state, angiogenic activity, and nuclear factor-kappa B (NF-κB) activation were assessed by immunohistochemical staining. The number, size, and level of inflammatory cytokines (Vegf, Il-6, Ccl-2, Lif) gene expression in the murine endometriosis-like lesions were reduced by BV6 treatment. BV6 repressed the intensity and rate of positive cells of CD3, F4/80, and PECAM immunostaining; in addition, the expression of NF-κB p65 and phospho-NF-κB p65 was also attenuated. Inhibitor of apoptosis proteins antagonist represses the inflammation status of murine endometriosis-like lesions viaNF-κB pathway. IAPs may be a novel therapeutic target for endometriosis.

Differential expression of EWI-2 in endometriosis, its functional role and underlying molecular mechanisms.

We aimed to investigate EWI-2 expression in endometrium tissues collected from women with endometriosis at mRNA and protein levels, to evaluate its potential as a biomarker for endometriosis and to study its functional role via possible regulation of the PI3K/Akt signaling pathway. Endometrium tissues were collected from patients with endometriosis and healthy individuals. EWI-2 mRNA expression was evaluated using quantitative real-time PCR (qRT-PCR) while EWI-2 protein levels were determined by western blotting. For functional studies, EWI-2 shRNA was transfected in endometrial epithelial cells and the in vitro migration and invasion assays were performed using the Transwell chambers. EWI-2 was significantly downregulated in tissues obtained from patients with endometriosis compared with healthy individuals (P < 0.0001). EWI-2 expression in the secretory phase was lower than that in the proliferative phase (P < 0.0001). Receiver-operator curve analysis of EWI-2 expression showed that the area under the curve for endometriosis diagnosis was 0.8942 (P = 0.003), 0.9643 (P = 0.0001), 0.9912 (P < 0.0001), and 0.9150 (P < 0.0001), respectively, for healthy women compared with women with endometriosis in matched comparisons of data originated from the proliferative, early, middle, and late secretory phases. Over the menstrual cycle, the expression of EWI-2 was significantly decreased in the eutopic tissues compared to the ectopic tissues. Further cellular and molecular analyses showed that EWI-2 inhibited cell migration and invasion via the Akt signaling. Our findings suggested that downregulation of EWI-2 may contribute to endometriosis physiopathology and potentiate EWI-2 as a valuable diagnostic biomarker and therapeutic target for endometriosis.

Expression, regulation and function of MicroRNAs in endometriosis.

Endometriosis (EMS), characterized by the presence and growth of functional en do met rial-like tissues outside the uterine cavity, is a common and benign gyneco logical disorder with a poorly understood and somewhat enigmatic etiopathogenesis and pathophysiology. MicroRNAs (miRNAs) are single-stranded 19-25 nucleotide-long RNAs and have an important role in post-transcriptional gene silencing by base pairing with target mRNAs. Recent research has shown that miRNAs and their target mRNAs are differentially expressed in endometriosis and other disorders of the female reproductive system. In this paper, we review the recent progress in understanding the roles of miRNAs in endometriosis, and specific miRNAs as biomarkers and therapeutic targets for endometriosis.

Hypoxia-inducible factor-1alpha: A promising therapeutic target in endometriosis.

Endometriosis is a common gynecologic disease defined as the presence of ectopic endometrial tissues on the ovaries and pelvic peritoneum, and it is a significant cause of pelvic pain, dysmenorrhea and infertility of women in their reproductive age. However, the etiology of endometriosis remains obscure. In recent years, a growing body of evidence validated that hypoxia developed a close relationship with endometriosis and the expression of hypoxia-inducible factor-1alpha (HIF-1α) was increased significantly in the development of endometriosis. Furthermore, inhibiting the expression of HIF-1α contributed to suppress endometriosis progression, suggesting HIF-1α plays a critical function in endometriosis. Nevertheless, the mechanisms by which HIF-1α associates with endometriosis are still undefined. In this brief review, we had a general understanding of HIF-1α firstly, and then we tried to sum up the collective knowledge of HIF-1α in endometriosis. Finally, we will discuss kinds of novel therapeutic approaches to endometriosis based on the functions of HIF-1α.

Angiogenesis as a therapeutic target in endometriosis.

Angiogenesis is a key factor for the successful establishment and growth of endometriotic lesions. We performed a literature search in PubMed and reviewed the most pertinent studies published until January 2014 and focused on the endometriosis-associated angiogenesis and/or anti-angiogenic strategies for the treatment of this gynecological disorder. The present review provides a concise summary of the known molecular mechanisms that promote vascularization of endometriotic lesions and may serve as potential therapeutic targets. We also present a systematic overview of the inclusive and exclusive anti-angiogenic agents that have been already studied in cell cultures, animal models and/or endometriosis patients. The integration of anti-angiogenic approaches in the multimodal management strategies for endometriosis patients will be conditioned by the outcomes of future assessments regarding the effectiveness of such treatments, the risk of drug resistance development and the incidence of unacceptable side effects.

Src as a novel therapeutic target for endometriosis

BackgroundEndometriosis is a common condition that is associated with an increased risk of developing ovarian carcinoma. Improved in vitro models of this disease are needed to better understand how endometriosis, a benign disease, can undergo neoplastic transformation, and for the development of novel treatment strategies to prevent this progression. MethodsWe describe the generation and in vitro characterization of novel TERT immortalized ovarian endometriosis epithelial cell lines (EEC16-TERT). ResultsExpression of TERT alone was sufficient to immortalize endometriosis epithelial cells. TERT immortalization induces an epithelial-to-mesenchymal transition and perturbation in the expression of genes involved in the development of ovarian cancer. EEC16-TERT was non-tumorigenic when xenografted into immunocompromised mice but grew in anchorage-independent growth assays in an epidermal growth factor and hydrocortisone dependent manner. Colony formation in agar was abolished by inhibition of Src, and the Src pathway was found to be activated in human endometriosis lesions. ConclusionsThis new in vitro model system mimics endometriosis and the early stages of neoplastic transformation in the development of endometriosis associated ovarian cancer. We demonstrate the potential clinical relevance of this model by identifying Src activation as a novel pathway in endometriosis that could be targeted therapeutically, perhaps as a novel strategy to manage endometriosis clinically, or to prevent the development of endometriosis-associated ovarian cancer.

The Cellular Inhibitor of Apoptosis Protein‐2 is a Possible Target of Novel Treatment for Endometriosis

How is the tumor necrosis factor (TNF) α-induced inhibitor of apoptosis (IAP) protein expression in endometriotic stromal cells (ESCs) involved in cell viability and signaling pathways? Endometriotic stromal cells were isolated from ovarian chocolate cysts in 20 patients who underwent laparoscopic surgery. IAP protein expression and IκB phosphorylation were evaluated by Western blot analysis. Interleukin (IL)-8 protein expression and cell proliferation were assessed by ELISA. Cellular IAP (cIAP)-2 protein expression in endometriotic tissue was higher than that of endometrium. TNFα markedly enhanced cIAP-2 protein expression in ESCs. Pretreatment with a nuclear factor (NF)-κB inhibitor attenuated TNFα-induced cIAP-2 expression. An antagonist of IAPs abrogated TNFα-induced cIAP-2 protein expression and showed a decrease in TNFα-induced IL-8 protein expression and BrdU incorporation in ESCs. TNFα and its downstream NFκB pathway have proven to be critical regulators of highly expressed cIAP-2 in ESCs. cIAP-2 may be a novel therapeutic target for endometriosis.

Histone deacetylase inhibitors down-regulate G-protein-coupled estrogen receptor and the GPER-antagonist G-15 inhibits proliferation in endometriotic cells

To investigate whether histone deacetylase inhibitors reduce the expression of the G-protein-coupled estrogen receptor (GPER) and whether the functional inhibition of GPER by the antagonist G-15 decreases the proliferation of endometriotic cells. Invitro study. University hospital. Immortalized epithelial endometriotic cells. Treatment with the histone deacetylase inhibitor romidepsin or suberoylanilide hydroxamic acid (SAHA), or with the GPER antagonist G-15. Western blot analysis and quantitative real-time polymerase chain reaction (PCR) were used to monitor the expression of GPER in response to drug treatment. Effects of GPER stimulation and inhibition on cell proliferation were investigated by the 93-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (Sigma) (MTT) assay. Our results demonstrate that romidepsin and SAHA reduce GPER expression in a concentration-dependent manner. This reduction correlated with the accumulation of acetylated histones. No decreased expression of the estrogen receptor (ER)-α and ERβ was found under comparable experimental conditions. Pretreatment of endometriotic cells with the GPER agonist G-1 stimulated cell proliferation accompanied by rapid Akt phosphorylation. G-15 reversed this stimulation and inhibited cell proliferation, which was accompanied by Akt dephosphorylation. G-protein-coupled estrogen receptor is proposed as a potential therapeutic target in endometriosis. The down-regulation of GPER and/or the impairment of its function may reduce the estrogen responsiveness in endometriosis, and therefore might be considered a possible treatment option of endometriosis.

MAPK signaling pathway and endometriosis: what is the link?

Endometriosis is defined as the presence of endometrial tissue outside the uterus, predominantly on the ovary and pelvic peritoneum [1]. It occurs in approximately 10 % of women of reproductive age and, although it is a benign disease, it causes infertility and pelvic pain, compromising the quality of life [1]. Out of various theories put forward to explain the origin of endometriosis, retrograde menstrual reflux has gained the widest acceptance [1, 2]. Endometriosis has been reported to be characterized by the presence of endometrial cells with capacity to avoid apoptosis beyond the uterine cavity [1, 2]. Apoptosis plays an important role in maintaining tissue homeostasis by striking a balance between proliferation and cell death [1]. Endometriotic cells have been described to show the peculiar biological characteristics of resistance to apoptosis with the inability to transmit apoptotic signal and the ability to avoid cell death [1, 2]. Recently, endometriosis has been correlated with aberrant endometrial expression of telomerase and survivin implying modifications in cell fate [1, 2]. Telomerase is a specialized transcriptase that can prevent telomere shortening [2, 3]. Telomeres are noncoding tandemly repeated DNA sequences that are vital for maintaining chromosomal integrity and cell stability [2]. The critical shortening of telomeres is linked to cell division and their senescence and death [2, 3]. Consequently, telomerase activation allows cells to overcome apoptosis acquiring immortal capacity [2, 3]. Survivin is the smallest member of the inhibitor of apoptosis protein family that acts by suppressing primarily caspases [1, 4]. It is prominently expressed in embryonic and fetal tissues and over-expressed in virtually all tumor types [1]. It is transcriptionally silent in most differentiated adult tissues, but it is expressed in ovary and in the proliferative phase of the cycling human endometrium, suggesting a physiological role in normal endometrial function [1, 4]. Survivin has been shown to be closely linked to escape from apoptosis of endometriotic cells and their viability with a critical role in the pathogenesis and progression of endometriosis together with telomerase [1–4]. It has been found that both the survivin and telomerase are up-regulated by epidermal growth factor (EGF) through MAPK (mitogen activated protein kinase) signaling pathway activation [3, 4]. MAPK has been reported to play an important role in many cellular reactions such as apoptosis, proliferation and inflammation [5]. Many studies have demonstrated that MAPK is involved directly in regulating the pathogenesis of endometriosis [5]. MAPK pathways seem to play a pivotal role as intracellular and extracellular signal transducers in endometriotic cells [6, 7]. It has been reported that enhanced proliferation and survival of eutopic endometrial cells from patients with endometriosis compared with healthy women have correlated with abnormal activation of MAPK signaling pathways [7]. In addition, accumulating evidences, show that MAPK pathways regulate pain hypersensitivity in different injury conditions implying their possible involvement in endometriosisrelated pelvic pain [8]. With respect to the above, we suggest that inhibition of MAPK signaling pathway downregulating both telomerase and survivin expression, may be candidate as a new therapeutic target for endometriosis. Moreover, understanding the possible regulatory role of MAPK in apoptosis and, ultimately, in the signal R. Mormile (&) Division of Pediatrics and Neonatology, Moscati Hospital, Via A. Gramsci, 3, 81031 Aversa, Italy e-mail: raffaellamormile@alice.it; raffaellamormile@libero.it

Inhibitor of apoptosis protein (IAP)-2 is a novel therapeutic target for endometriosis

We previously showed that gene and protein of four Inhibitor of Apoptosis Protein (IAP) family (cIAP-1, cIAP-2, XIAP, and survivin) in endometriotic tissues were highly expressed compared with those in eutopic endometrial tissues. We also revealed that concentrations of TNFα and IL-8 are elevated in peritoneal fluid of women with endometriosis, and that IL-8 enhanced proliferation of endometriotic stromal cells via NFκB activation. we focused on the regulation of IAP family expression in endometriotic stromal cells and further aimed to delineate the mechanism involved in pathogenesis of endometriosis.

Proteinase-activated receptors in the endometrium and endometriosis

Proteinase-activated receptors (PARs) are G protein-coupled receptors activated by various proteinases. PARs play important roles in haemostasis, thrombosis, and inflammation. PAR₁ and PAR₂ are expressed in endometrial cells from the eutopic endometrium and endometriotic cells derived from endometriotic lesions. A typical activator of PAR₁, thrombin, and a typical activator of PAR₂, tryptase, are produced in the endometrium as well as endometriotic lesions. PAR₁ activation in endometrial stromal cells induces production of vascular endothelial growth factor and matrix metalloproteinases, and increases activities of tissue-type and urokinase-type plasminogen activator. PAR₂ activation in endometrial stromal cells stimulates interleukin (IL)-8 and stem cell factor production and proliferation of the cells. PAR₁ activation in endometriotic stromal cells induces production of IL-8, monocyte chemotactic protein-1, and cyclooxygenase-2, and proliferation of the cells. PAR₂ activation in endometriotic stromal cells increases secretion of IL-6 and IL-8, and the number of the cells. These findings indicate a wide range of function of PAR₁ and PAR₂ in the endometrium and endometriosis, and suggest PAR₁ and PAR₂ as possible therapeutic targets for endometriosis.

Proteinase-activated receptors in the endometrium and endometriosis

Proteinase-activated receptors (PARs) are G protein-coupled receptors activated by various proteinases. PARs play important roles in haemostasis, thrombosis, and inflammation. PAR₁ and PAR₂ are expressed in endometrial cells from the eutopic endometrium and endometriotic cells derived from endometriotic lesions. A typical activator of PAR₁, thrombin, and a typical activator of PAR₂, tryptase, are produced in the endometrium as well as endometriotic lesions. PAR₁ activation in endometrial stromal cells induces production of vascular endothelial growth factor and matrix metalloproteinases, and increases activities of tissue-type and urokinase-type plasminogen activator. PAR₂ activation in endometrial stromal cells stimulates interleukin (IL)-8 and stem cell factor production and proliferation of the cells. PAR₁ activation in endometriotic stromal cells induces production of IL-8, monocyte chemotactic protein-1, and cyclooxygenase-2, and proliferation of the cells. PAR₂ activation in endometriotic stromal cells increases secretion of IL-6 and IL-8, and the number of the cells. These findings indicate a wide range of function of PAR₁ and PAR₂ in the endometrium and endometriosis, and suggest PAR₁ and PAR₂ as possible therapeutic targets for endometriosis.

Proteinase-activated receptors in the endometrium and endometriosis

Proteinase-activated receptors (PARs) are G protein-coupled receptors activated by various proteinases. PARs play important roles in haemostasis, thrombosis, and inflammation. PAR1 and PAR2 are expressed in endometrial cells from the eutopic endometrium and endometriotic cells derived from endometriotic lesions. A typical activator of PAR1, thrombin, and a typical activator of PAR2, tryptase, are produced in the endometrium as well as endometriotic lesions. PAR1 activation in endometrial stromal cells induces production of vascular endothelial growth factor and matrix metalloproteinases, and increases activities of tissue-type and urokinase-type plasminogen activator. PAR2 activation in endometrial stromal cells stimulates interleukin (IL)-8 and stem cell factor production and proliferation of the cells. PAR1 activation in endometriotic stromal cells induces production of IL-8, monocyte chemotactic protein-1, and cyclooxygenase-2, and proliferation of the cells. PAR2 activation in endometriotic stromal cells increases secretion of IL-6 and IL-8, and the number of the cells. These findings indicate a wide range of function of PAR1 and PAR2 in the endometrium and endometriosis, and suggest PAR1 and PAR2 as possible therapeutic targets for endometriosis.

Proangiogenic Tie2 + Macrophages Infiltrate Human and Murine Endometriotic Lesions and Dictate Their Growth in a Mouse Model of the Disease

Endometriosis affects women of reproductive age, causing infertility and pain. Although immune cells are recruited in endometriotic lesions, their role is unclear. Tie2-expressing macrophages (TEMs) have nonredundant functions in promoting angiogenesis and growth of experimental tumors. Here we show that human TEMs infiltrate areas surrounding newly formed endometriotic blood vessels. We set up an ad hoc mouse model in which TEMs, and not Tie2-expressing endothelial cells, are targeted. We transplanted in wild-type recipients bone marrow cells expressing a suicide gene (Herpes simplex virus type 1 thymidine kinase) under the Tie2 promoter/enhancer. TEMs infiltrated endometriotic lesions. TEM depletion by ganciclovir administration arrested the growth of established lesions, without toxicity. Lesion architecture was disrupted, with: i) loss of glandular organization, ii) reduced neovascularization, and iii) activation of caspase 3 in CD31(+) endothelial cells. Thus, TEMs are important for maintaining the viability of newly formed vessels and represent a potential therapeutic target in endometriosis.

Expression and possible role of non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) in the human endometrium and endometriosis

Non-steroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1) is involved in cellular processes such as inflammation, apoptosis and tumorigenesis. However, little is known about the expression and function of NAG-1 in the endometrium. This study aimed to evaluate the expression of NAG-1 in the endometrium and in the absence or presence of endometriosis and to investigate the effect of celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, on NAG-1 mRNA levels and apoptosis in human endometrial stromal cells (HESCs). Eutopic endometrial samples were obtained during surgery from 40 patients with, and 40 patients without, endometriosis. Real-time PCR was used to quantify NAG-1 mRNA levels and immunohistochemistry was used to localize NAG-1 protein in the endometrium. To investigate the effects of celecoxib, HESCs were isolated and cultured with different concentrations of celecoxib or with 100 µM celecoxib at different times. Apoptosis was assessed by flow cytometry. NAG-1 mRNA levels and immunoreactivity showed cyclical changes through the menstrual cycle, increasing during the late secretory and menstrual phases. NAG-1 mRNA and protein levels were significantly lower in patients with endometriosis, compared with the control group. Celecoxib induced NAG-1 mRNA levels and apoptosis in cultured HESCs, with the effects dependent on drug concentrations and duration of treatment. Celecoxib treatment had no effect on prostaglandin E(2) levels in the culture supernatants. NAG-1 may be important in maintaining homeostasis in the normal endometrium and alterations in NAG-1 expression may be associated with the establishment of endometriosis. NAG-1 might be a therapeutic target for endometriosis.

Nuclear factor-kappa B is constitutively activated in peritoneal endometriosis

Red (active), black and white endometriotic lesions are characteristic of peritoneal endometriosis. The transcription factor nuclear factor-kappa B (NF-kappaB) activates proinflammatory, proliferative and antiapoptotic genes in many cell types. To determine whether NF-kappaB is activated in peritoneal endometriosis in women, and further ascertain the differential inflammatory status of endometriotic implants, NF-kappaB activation and intercellular adhesion molecule (ICAM)-1 expression were investigated in peritoneal endometriotic lesions according to their type. Furthermore, p65 and p50 subunits of active NF-kappaB dimers were evaluated in endometriotic lesions to gain some insight into NF-kappaB-implicated pathways. Thirty-six biopsies of peritoneal endometriotic lesions were analyzed. Constitutive NF-kappaB activation, involving p65- and p50-containing dimers, was demonstrated in peritoneal endometriotic lesions by electrophoretic mobility shift assays and supershift analyses, as well as NF-kappaB (p65) DNA-binding activity immunodetection assays. NF-kappaB activation and ICAM-1 expression (evaluated by immunoblotting) were significantly higher in red lesions than black lesions, whereas IkappaBalpha (NF-kappaB inhibitory protein) expression was constant, as shown by western blot analysis. This is the first study to demonstrate constitutive NF-kappaB activation in peritoneal endometriosis in women. NF-kappaB activation and ICAM-1 expression in red lesions confirm the more extensive inflammatory pattern of these lesions compared with black lesions. The involvement of p50/p65 dimers in NF-kappaB activation suggests implication of the classic NF-kappaB activation pathway, making it an attractive therapeutic target in endometriosis.

Endometriosis-Specific Genes Identified by Real-Time Reverse Transcription-Polymerase Chain Reaction Expression Profiling of Endometriosis Versus Autologous Uterine Endometrium

The etiology and molecular pathogenesis of endometriosis, a prevalent estrogen-dependent gynecologic disease, are poorly understood. The objective of the study was to identify the differentially expressed genes between autologous ectopic and eutopic endometrium. Subtractive hybridization was used for a genome-wide search for differentially expressed genes between autologous ectopic and eutopic endometrium. Real-time RT-PCR was used for gene expression profiling in the paired tissue samples taken from multiple subjects. The paired pelvic endometriosis and uterine endometrium tissue biopsies were procured from 15 patients undergoing laparoscopy or hysterectomy for endometriosis. Seventy-eight candidate genes were identified from the subtractive cDNA libraries. Seventy-six of these genes were investigated in approximately 8000 real-time PCR for their differential expression in 30 paired tissue biopsies from 15 patients affected by endometriosis. Cluster analysis on gene expression revealed highly consistent profiles in two groups of genes, despite the clinical heterogeneity of the 15 cases. Thirty-four genes specific to early disease point to their potential roles in establishment and evolution of endometriosis. Most interestingly, 14 genes were consistently dysregulated in the paired samples from the majority of the patients. Of these, there were two uncharacterized transcripts and two novel genes, and 10 were matched to known genes: IGFBP5, PIM2, RPL41, PSAP, FBLN1, SIPL, DLX5, HSD11B2, SET, and RHOE. Dysregulation of 14 genes was found to be overtly associated with endometriosis. Some of these genes, known to participate in estrogen activities and antiapoptosis, may play a role in the pathogenesis of endometriosis and may represent potential diagnostic markers or therapeutic targets for endometriosis.