Leiomyoma Cells Research Articles

8636 Characterization Of The Role Of Neutrophils In Promoting Progression Of Lymphangioleiomyomatosis

Abstract Disclosure: T.R. Henson: None. B. Minor: None. S.R. Hammes: None. Lymphangioleiomyomatosis (LAM) is a rare, multisystemic disease that is associated with cystic lung destruction. LAM is caused by small smooth muscle cell-like tumors throughout the lung that contain deleterious mutations in the tuberous sclerosis complex genes TSC1 or TSC2. This attenuates the activity of the mammalian target of rapamycin complex 1 (mTORC1) causing increased proliferation of the smooth muscle cells. LAM is a sexually dimorphic disease that primarily affects genetic females. Clinical observations implicate the role of estrogen in disease progression, as it is exacerbated during pregnancy and by use of exogenous estrogens in birth control. LAM tumor-derived cells lines have shown that direct estrogen stimulation of cell proliferation is modest compared to the dramatic in vivo effects of estradiol (E2) stimulation in a uterine-specific TSC2-null mouse model. Creation of a monoclonal cell line from these mice resulted in the generation of the LTM3 cell line, which also demonstrates low response to direct estrogen stimulation in vitro. This indicates that direct E2 stimulation may act upon other cell types in the tumor microenvironment. Immunophenotyping of uterine-specific TSC2-null mice has shown that the tumor microenvironment is enriched with myeloid cells specifically neutrophils. Functional analysis of neutrophil suppression in vivo has shown a reduction in myometrial growth in the uterine-specific TSC2-null mice, indicating a key role for neutrophil-mediated LAM tumor growth. Altered transcriptional regulation of neutrophils further highlights the importance of neutrophils in LAM progression. Bulk messenger RNA sequencing revealed E2 dependent increase in the neutrophil derived serine protease neutrophil elastase (NE). NE in vitro has been shown to increase migration, invasion, and proliferation of LAM cell lines, while inhibition in vivo has shown a reduction to the myometrial growth. Previously published data by the lab has shown that E2 increases tumor induced expansion of neutrophils in the bone marrow. Isolation and purification of Neutrophils from mouse bone marrow stimulated with tumor conditioned media from the LTM3 cells has shown signs of activation via upregulated gene expression. The focus of the current research has been the characterization of bone marrow derived neutrophils. Currently working with an in vitro system to study the proliferation of LTM3 and the rat leiomyoma (ELT3) cell lines when they are co-cultured with neutrophils. Understanding the mechanism by which neutrophils are activated and interact with LAM cells can give understanding to disease progression may provide insight into future treatment options. Presentation: 6/3/2024

Coenzyme Q-10 reduced the aberrant production of extracellular matrix proteins in uterine leiomyomas through transforming growth factor beta 3

To evaluate the impact of coenzyme Q-10 (CoQ-10) on the dysregulated synthesis of extracellular matrix proteins mediated by transforming growth factor beta 3 (TGF-β3) in uterine leiomyomas. Laboratory study. University. None. Treatment of immortalized uterine myometrial and leiomyoma cells to TGF-β3 and CoQ-10. The protein concentrations of collagen 1A1 (COL1A1), collagen 3A1 (COL3A1), collagen 11A1 (COL11A1), and fibronectin (FN1) were assessed through western blot analysis after treatment of immortalized uterine myometrial and leiomyoma cells with both transforming growth factor beta (TGF-β) 3 and concentrations of CoQ-10 at 10, 50, and 100 μM concurrently for 24 hours. Immortalized uterine leiomyoma and myometrial cells exposed to TGF-β3 for 24 hours demonstrated a significant up-regulation of COL1A1, COL3A1, COL11A1, and FN1 compared with untreated cells. In leiomyoma cells, concurrent treatment with CoQ-10 over the same timeframe revealed a dose-dependent decrease in these protein concentrations compared with those in cells treated with TGF-β3 alone. At the highest concentration of 100 μM of CoQ-10, significant decreases in the amounts of COL1A1 (0.59 ± 0.10-fold), COL3A1 (0.46 ± 0.09-fold), COL11A1 (0.53 ± 0.09-fold), and FN1 (0.56 ± 0.09-fold) were observed. Similarly, myometrial cells exposed to both TGF-β3 and CoQ-10 demonstrated a dose-responsive decline in the amount of extracellular matrix protein compared with cells exposed to TGF-β3 alone. Significant reductions in the amounts of COL1A1 (0.75 ± 0.03-fold), COL3A1 (0.48 ± 0.06-fold), COL11A1 (0.38 ± 0.06), and FN1 (0.69 ± 0.04-fold) were appreciated at 100-μM CoQ-10. Coenzyme Q-10 mitigated the aberrant production of key biomarkers of the extracellular matrix mediated by TGF-β3 in uterine leiomyomas. Our findings highlight a promising nonhormonal compound that can counteract the fibroproliferative process inherent to leiomyomas.

HMGA2 overexpression induces plasticity in myometrial cells and a transcriptomic profile more similar to that of uterine fibroids

ObjectiveTo study the possible role for HMGA2 overexpression in differentiated myometrial cells and its potential to induce a stem cell-like or dedifferentiating phenotype and drive fibroid development. DesignMyometrial cells were immortalized and transduced with an HMGA2 lentivirus to produce HMGA2hi cells. In vitro stem cell assays were conducted and RNA from HMGA2hi and control cells and fibroid-free myometrial (MyoN) and HMGA2 fibroid (HMGA2F) tissues were submitted for RNA-sequencing. SettingUniversity research laboratory SubjectsWomen undergoing hysterectomy for symptomatic uterine fibroids or other gynecological conditions. InterventionNot applicable. Main Outcome MeasuresIn-vitro stem-like properties from myometrium cell lines. RNA-sequencing and collagen production of HMGA2 overexpressing primary leiomyoma tissue and cell lines. ResultsHMGA2hi cells have enhanced self-renewal capacity, decreased proliferation, and have a greater ability to differentiate into other mesenchymal cell types. HMGA2hi cells exhibit a stem cell-like signature and share transcriptomic similarities with HMGA2F. Moreover, dysregulated extracellular matrix pathways are observed in both HMGA2hi cells and HMGA2F. ConclusionOur findings suggest that HMGA2 overexpression drives myometrial cells to dedifferentiate into a more plastic phenotype and provides evidence for an alternative mechanism for fibroid etiology, suggesting that fibroids may not only arise from a mutated stem cell but also from a mutated differentiated myometrial cell.

Spectrum of endoscopic gastric subepithelial lesions encountered on EUS-FNA: A single center experience.

Endoscopic ultrasound guided fine needle aspiration (EUS-FNA) is a minimally invasive and reliable non-surgical technique for the diagnosis of gastrointestinal lesions. The present study aimed to evaluate the spectrum of lesions encountered in the gastric subepithelium on EUS-FNA at a tertiary care center. Archival data of all patients undergoing EUS-FNA for gastric submucosal lesions over a period of 5 years was retrieved. Patient demographics, clinical presentation, and EUS findings were recorded along with the FNA results. A total of 78 EUS-FNA samples were analyzed. Material was adequate in 62 cases (79.48%) and inadequate in 16 cases (12.82%) patients due to scant cellularity. Of the adequate samples, 34 (43.5%) were reported as neoplastic while 20 (25.64%) were non-neoplastic, and 8 (10.25%) were reported as suspicious of a neoplasm. In the neoplastic category, the predominant diagnosis was of spindle cell neoplasm comprising gastrointestinal stromal tumor (13), benign neural tumor (03), leiomyoma (02), and spindle cell tumors (03). The latter could not be categorized further due to a lack of IHC material. The next common diagnosis was adenocarcinoma (06) followed by neuroendocrine tumor (02) and poorly differentiated carcinoma (01). The non-neoplastic lesions included non-specific pathology (15), inflammatory lesions (08), and one case each of tuberculosis, pancreatic rest, and Brunner gland hamartoma. Cell blocks for ancillary testing were available in 54 cases (65.23%) and follow-up was available in 42 cases (53.84%). EUS-FNA is a good modality for the diagnosis of gastric submucosal lesions with a high diagnostic yield.

Differential effect of estrogen and progesterone on <i>in vitro</i> growth of uterine leiomyoma cells with chromosome 7 deletions

BACKGROUND: The studies on how sex steroid hormones affect growth of uterine leiomyoma cells with chromosomal abnormalities is highly relevant for development of personalized tumor therapy.
 AIM: To study in vitro the isolated and combined effects of estrogen and progesterone on uterine leiomyoma cells with chromosomal aberrations — deletions in 7q.
 MATERIALS AND METHODS: The study was performed on 15 uterine leiomyomas, excised from 15 women of 26–44 years of age who were not treated with hormones. Uterine leiomyoma cells were cultured in hormone-free medium, in the medium supplemented with estrogen, progesterone or both hormones. The chromosome preparations were made and stained with QFH/AcD to perform conventional karyotyping and fluorescence in situ hybridization (FISH) to accurately describe chromosomal rearrangements. The frequency of uterine leiomyoma cells with chromosomal aberrations was assessed by interphase FISH.
 RESULTS: Deletions in 7q were identified in 6 out of 15 karyotyped uterine leiomyomas; four of them had one clone with deletion in 7q whereas two others comprised two clones with 7q deletions of different length. The frequency of cells carrying deletions in 7q greatly varied in uterine leiomyoma samples cultured in hormone-free medium: from 3.5% to 93.6%. Exposure of cell cultures to estrogen and progesterone resulted in a fold change frequency increase in some of the uterine leiomyomas and decrease in the others. The most significant changes in the frequency of cells with deletions in 7q were registered in response to the isolated estrogen and, to a lesser extent, to progesterone exposure; less significant changes were observed after combined hormonal effect.
 CONCLUSIONS: In uterine leiomyomas with deletions in 7q, the frequency of abnormal cells may either increase or decrease in response to estrogen and progesterone in vitro supplementation. The isolated effect of estrogen or progesterone on the frequency of uterine leiomyoma cells with deletion in 7q is more pronounced compared to the combined one.

Simvastatin induces degradation of the extracellular matrix in human leiomyomata: novel in vitro, in vivo, and patient level evidence of matrix metalloproteinase involvement

ObjectivesTo assess the effect of simvastatin on uterine leiomyoma growth and extracellular matrix (ECM) deposition. DesignLaboratory analysis of human leiomyoma cell culture, xenograft in a mouse model, and patient tissue from a clinical trial. SettingAcademic research center. Patient(s)Tissue culture from human leiomyoma tissue and surgical leiomyoma tissue sections from a placebo-controlled randomized clinical trial. Intervention(s)Simvastatin treatment. Main Outcome Measure(s)Serum concentrations, xenograft volumes, and protein expression. ResultsMice xenografted with 3-dimensional human leiomyoma cultures were divided as follows: 7 untreated controls, 12 treated with activated simvastatin at 10 mg/kg body weight, and 15 at 20 mg/kg body weight. Simvastatin was detected in serum from mice injected at the highest dose. Xenograft volumes were significantly smaller (mean 53% smaller at the highest concentration, p < 0.05). There was dissolution of compact ECM, decreased ECM formation, and lower collagen protein expression in xenografts. Membrane type 1 matrix metalloproteinase (MT1-MMP) was increased in vitro and in vivo. Matrix metalloproteinase 2 (MMP2) and low density lipoprotein receptor related protein 1 (LRP1) were increased in vitro. ConclusionsSimvastatin exhibited antitumoral activity, with extracellular matrix degradation and decreased leiomyoma tumor volume in vivo. Activation of the MMP2/MT1-MMP/LRP1 pathway may explain these findings.

Novel Approaches to Possible Targeted Therapies and Prophylaxis of Uterine Fibroids.

Uterine leiomyomas are the most common benign tumors in women of childbearing age. They may lead to problems of conception or complications during the gestational period. The methods of treatment include surgical (myomectomy and hysterectomy, embolization of arteries) and therapeutic treatment (ulipristal acetate, leuprolide acetate, cetrorelix, goserelin, mifepristone). Both approaches are efficient but incompatible with pregnancy planning. Therefore, there is a call for medical practice to develop therapeutical means of preventing leiomyoma onset in patients planning on becoming pregnant. Based on the analysis of GWAS data on the search for mononucleotide polymorphisms associated with the risk of leiomyoma, in meta-transcriptomic and meta-methylomic studies, target proteins have been proposed. Prospective therapeutic treatments of leiomyoma may be based on chemical compounds, humanized recombinant antibodies, vaccines based on markers of the uterine leiomyoma cells that are absent in the adult organism, or DNA and RNA preparations. Three different nosological forms of the disease associated with driver mutations in the MED12, HMGA2, and FH genes should be considered when developing or prescribing drugs. For example, synthetic inhibitors and vaccines based on matrix metalloproteinases MMP11 and MMP16 are expected to be effective only for the prevention of the occurrence of MED12-dependent nodules.

MED12 mutation activates the tryptophan/kynurenine/AHR pathway to promote growth of uterine leiomyomas.

Uterine leiomyomas cause heavy menstrual bleeding, anemia, and pregnancy loss in millions of women worldwide. Driver mutations in the transcriptional mediator complex subunit 12 (MED12) gene in uterine myometrial cells initiate 70% of leiomyomas that grow in a progesterone-dependent manner. We showed a distinct chromatin occupancy landscape of MED12 in mutant MED12 (mut-MED12) versus WT-MED12 leiomyomas. Integration of cistromic and transcriptomics data identified tryptophan 2,3-dioxygenase (TDO2) as the top mut-MED12 target gene that was significantly upregulated in mut-MED12 leiomyomas when compared with adjacent myometrium and WT-MED12 leiomyomas. TDO2 catalyzes the conversion of tryptophan to kynurenine, an aryl hydrocarbon receptor (AHR) ligand that we confirmed to be significantly elevated in mut-MED12 leiomyomas. Treatment of primary mut-MED12 leiomyoma cells with tryptophan or kynurenine stimulated AHR nuclear translocation, increased proliferation, inhibited apoptosis, and induced AHR-target gene expression, whereas blocking the TDO2/kynurenine/AHR pathway by siRNA or pharmacological treatment abolished these effects. Progesterone receptors regulated the expression of AHR and its target genes. In vivo, TDO2 expression positively correlated with the expression of genes crucial for leiomyoma growth. In summary, activation of the TDO2/kynurenine/AHR pathway selectively in mut-MED12 leiomyomas promoted tumor growth and may inform the future development of targeted treatments and precision medicine.

The combination of natural compounds Crila and epigallocatechin gallate showed enhanced antiproliferative effects on human uterine fibroid cells compared with single treatments

To investigate the combined effects of Crila and green tea extract, epigallocatechin gallate (EGCG), compared with single treatments, on human uterine fibroid cells. Human uterine leiomyoma (HuLM) cells were treated with different concentrations of Crila, alone or in combination with EGCG, and several experiments were employed. A laboratory study. N/A. Crila, EGCG. Cell proliferation assay, drug synergy using combination index, protein and gene expression analysis of proliferation marker proliferating cell nuclear antigen, and apoptosis marker BAX using western blotting and quantitative polymerase chain reaction, respectively. Results showed that tested Crila concentrations, when combined with 25 and 50 μM EGCG, exerted synergistic growth inhibitory effects on HuLM viability. This inhibitory effect on HuLM cell viability was because of decreased cell proliferation, as shown by a decrease in the proliferation marker proliferating cell nuclear antigen at messenger RNA and protein levels, rather than inducing apoptosis. Our study concludes that the utility of natural compounds may provide a safe and cost-effective alternative to currently used short-term hormonal therapies against uterine fibroids.

FTIR Microspectroscopy as a new probe to study human uterine lesions: Characterization of tumor cell lines from uterine smooth muscle cells and evaluation of EPA and DHA in vitro treatments

During their life, women are likely to develop uterine diseases, which often compromise their fertile and perimenopausal age. Besides benign lesions like leiomyomas, several malignant neoplasms can occur, such as the uterine leiomyosarcoma, which represents the most frequent malignancy among the rarest uterine cancers. It presents several variants similar to both benign and malignant neoplasms, and sometimes it shares symptoms with the benign counterpart. In this scenario, for a correct diagnosis and a successful prognosis, it is mandatory to detect new reliable markers which strengthen histopathological outcomes and let define a more appropriate and less harmful therapy. Based on this concerning evidence, in the present study, Fourier Transform Infrared Microspectroscopy has been exploited at a cellular level on uterine leiomyoma and leiomyosarcoma cell lines to (1) identify specific spectral biomarkers able to distinguish between benign and malignant lesions, and (2) evaluate the efficacy of eicosapentaenoic and docosahexaenoic acids (respectively EPA and DHA), already successfully tested. Results evidenced reliable differences in the spectral signature of benign and malignant cells, mainly in terms of lipids and nucleic acids composition. Moreover, even if EPA and DHA seemed to exert different effects on the tested cell lines, no cytotoxic and/or anti-apoptotic actions were observed after omega-3 based treatments.

Giant middle mediastinal lesions: when tumor size correlates with mesenchymal origin-a retrospective single-center analysis.

The International Thymic Malignancy Interest Group (ITMIG) proposed an internationally accepted division of the mediastinum into three compartments based on computed tomography (CT): anterior (prevascular), middle (visceral) and posterior (paravertebral) compartment. There is no generally accepted definition for the term "giant" when applied to middle mediastinal lesions. We defined the term "giant" and described our surgical experience in treating patients with giant lesions of the middle mediastinum. CT imaging of patients operated in our center from January 2016 to August 2021 for mediastinal lesions was reviewed. Lesions were categorized to one of the ITMIG-defined compartments. Lesion size at diagnosis was measured at its largest diameter on axial CT imaging. Giant middle mediastinal lesions were defined as lesions having a size ≥90th percentile of our middle mediastinal lesion cohort. Patients with giant middle mediastinal lesions were further analyzed. Thirty-six patients (23%) had lesions located in the middle mediastinal compartment. Most common diagnoses were mediastinal cysts (n=10, 28%), metastatic lesions (n=6, 17%), lymphomas (n=5, 14%), and sarcomas (n=3, 8%). Ninetieth percentile lesion size was 73 mm. As per definition, four patients had giant middle mediastinal lesions. All these four lesions were of mesenchymal origin including oesophageal leiomyoma, synovial sarcoma, leiomyosarcoma and undifferentiated round cell sarcoma. Resection was performed through posterolateral thoracotomy or sternotomy, with or without cardiopulmonary bypass. The term "giant" could be defined as a mass larger or equal to 73 mm. This definition selected specifically lesions with mesenchymal origin and may therefore guide diagnostic algorithm and patient management.

Oxidative stress mediates the inhibitory effects of Manzamine A on uterine leiomyoma cell proliferation and extracellular matrix deposition via SOAT inhibition

Uterine fibroids, the most common benign tumors of the myometrium in women, are characterized by abnormal extracellular matrix deposition and uterine smooth muscle cell neoplasia, with high recurrence rates. Here, we investigated the potential of the marine natural product manzamine A (Manz A), which has potent anti-cancer effects, as a treatment for uterine fibroids. Manz A inhibited leiomyoma cell proliferation in vitro and in vivo by arresting cell cycle progression and inducing caspase-mediated apoptosis. We performed target prediction analysis and identified sterol o-acyltransferases (SOATs) as potential targets of Manz A. Cholesterol esterification and lipid droplet formation were reduced by Manz A, in line with reduced SOAT expression. As a downstream target of SOAT, Manz A also prevented extracellular matrix deposition by inhibiting the β-catenin/fibronectin/metalloproteinases axis and enhanced autophagy turnover. Excessive free fatty acid accumulation by SOAT inhibition led to reactive oxygen species to impair mitochondrial oxidative phosphorylation and trigger endoplasmic reticulum stress via PERK/eIF2α/CHOP signaling. The inhibitory effect of ManzA on cell proliferation was partially restored by PERK knockdown and eliminated by tauroursodeoxycholic acid, suggesting oxidative stress plays a critical role in the mechanism of action of Manz A. These findings suggest that targeting SOATs by Manz A may be a promising therapeutic approach for uterine fibroids.

Increased Occurrence of Cutaneous Leiomyomas and Dermatofibromas in Patients with Uterine Leiomyomas without Fumarate Hydratase Gene Mutations.

Leiomyomas are smooth muscle-derived benign neoplasms that can affect all organs, most frequently in the uterus. Fumarate hydratase gene (FH) mutation is characterised by an autosomal dominant disease with increased occurrence of renal tumours, but also by cutaneous (CLs) and uterine leiomyomas (ULs). So far, an increased occurrence of skin tumours in non-mutated patients with ULs has not been verified. To this aim, a case-group of women who were FH non-mutated patients surgically treated for ULs (n = 34) was compared with a control-group (n = 37) of consecutive age-matched healthy women. The occurrence of skin neoplasms, including CLs and dermatofibromas (DFs), was evaluated. Moreover, the microscopic features of FH non-mutated skin tumours were compared with those of an age-matched population group (n = 70) who presented, in their clinical history, only one type of skin tumour and no ULs. Immunohistochemical and in vitro studies analysed TGFβ and vitamin D receptor expression. FH non-mutated patients with ULs displayed a higher occurrence of CLs and DFs (p < 0.03 and p < 0.001), but not of other types of skin tumours. Immunohistochemistry revealed a lower vitamin D receptor (VDR) expression in CLs and DFs from the ULs group compared with those from the population group (p < 0.01), but a similar distribution of TGFβ-receptors and SMAD3. In vitro studies documented that TGFβ-1 treatment and vitamin D3 have opposite effects on α-SMA, TGFβR2 and VDR expression on dermal fibroblast and leiomyoma cell cultures. This unreported increased occurrence of CLs and DFs in FH non-mutated patients with symptomatic ULs with vitamin D deficiency suggests a potential pathogenetic role of vitamin D bioavailability also for CLs and DFs.

Potential mechanism of Taohong Siwu Decoction in uterine fibroid treatment based on integrated strategy of network pharmacology and experimental verification

BackgroundTaohong Siwu Decoction (THSWD) is a widely prescribed Traditional Chinese Medicine (TCM) for treating gynecological diseases. It is used to treat uterine fibroids (UF) in China, while its potential therapeutic effects and mechanism are unknown.MethodsThe present study used network pharmacology to identify PI3K/AKT as one of the main THSWD signaling pathways that can be targeted to treat UF. The potential binding sites of miR-21-5p to PTEN were predicted using online databases. We were able to establish a UF rat model successfully. We selected the 15% THSWD serum after preparing THSWD drug-containing serum to culture tumor tissue-derived cells. These studies enabled us to assess the role of THSWD in UF improvement.ResultsIn vivo, we observed that low, medium, and high doses of THSWD improved histological changes in UF rats by increasing the expression levels of PTEN and miR-21-5p in their uterus while decreasing the expression levels of p-PI3K, p-AKT, and miR-21-5p. Treatment with THSWD medicated serum (15%) effectively inhibited the proliferation of cells derived from human UF and promoted apoptosis in vitro. PI3K phosphorylation, Akt phosphorylation, and miR-21-5p expression were decreased, while PTEN and cleaved caspase-3 were increased. These findings were reversed by administering 740 Y-P (a PI3K/Akt pathway agonist) and a miR-21-5p mimic. In addition, the double luciferase reporter gene assay confirmed the targeted binding relationship between miR-21-5p and PTEN.ConclusionsTHSWD inhibited the expression and activation of the PI3K/AKT and miR-21-5p/PTEN pathways, resulting in anti-UF activity in leiomyoma cell models. Our findings suggest that THSWD could be used to treat UF.

P-356 establishment of a novel 3D spheroid culture system of uterine leiomyoma cells

Abstract Study question To establish three-dimensional (3D) spheroid culture system of uterine leiomyoma (ULM) cells that responds to estrogen(E) and progesterone(P). Summary answer We established the 3D spheroid culture system of ULM cells, in which the ULM-spheroid grows in response to progesterone. What is known already ULMs proliferate in response to female hormones in the patients-derived xenograft model in vivo. However, it is controversial whether ULM cells proliferate in response to female hormones in in vitro monolayer culture system. We reported that ULM cells formed nodule-like aggregates of cells when the cells were grown in 3D culture using collagen gel. However, 3D spheroid culture system of ULM cells is not established yet. Recently, somatic mutations in the MED12 gene were detected in almost 70% of ULMs. It is also unclear whether responsiveness of ULM cells to female hormones differs between MED12-mutation positive and negative ULMs. Study design, size, duration Fourteen and 13 ULMs obtained from 18 premenopausal women were used for monolayer culture and 3D spheroid culture, respectively. The isolated ULM cells were confirmed to be smooth muscle cells with 80% purity and to have progesterone receptors by immunocytochemistry. The cells were preincubated for 2 days and then used for the following experiments below. MED12-mutation status was analyzed by Sanger sequencing. Monolayer and spheroid cultures were done on MED12-mutation positive and negative ULM cells. Participants/materials, setting, methods For the monolayer culture system, cells were incubated with E+P, E alone, P alone, and control (without E+P) for 7 days. For 3D culture system, cells were incubated in the low attachment well dish to induce floating cell culture, and after 24 h, we identified the floating cell aggregates as spheroid of ULM cells. Then, the ULM-spheroids were incubated with E+P, E alone, P alone, E+P+selective progesterone receptor modulator (SPRM), and control for 7 days. Main results and the role of chance Responsiveness to female hormones was assessed by counting cells for monolayer culture, and by measuring the cross-sectional area of the spheroid for 3D culture system. ULM cells did not proliferate by any female hormones in the monolayer culture system. In the 3D spheroid culture system, the ULM-spheroid cells showed expression of alpha-smooth muscle actin and vimentin, indicating that most of the spheroid cells are smooth muscle cells. In MED12-mutation negative ULMs, the mean of the cross-sectional area of the spheroid of E+P , P alone, E alone, E+P+SPRM and control were 0.375mm², 0.343mm², 0.237mm², 0.252mm² and 0.193mm², respectively, while in MED12-mutation positive ULMs, the area were 0.349mm², 0.338mm², 0.268mm², 0.264mm². and 0.215mm². The morphology of the spheroid of the E alone, E+P+SPRM and control groups showed the loss of smooth muscle cells. These results suggest that the growth of the ULM-spheroid is maintained by progesterone, and that the responsiveness of the spheroid to progesterone does not differ between MED-mutation negative and positive ULM cells. Our study showed that ULM cells of the 3D spheroid culture system acquire function to respond to progesterone. Limitations, reasons for caution Further studies are needed to investigate whether the 3D spheroid culture system established in this study accurately reflects the responsiveness to female hormones of the in vivo. Wider implications of the findings Our study showed that the 3D spheroid culture system of ULM cells has the responsiveness to progesterone whereas the monolayer culture system does not. The 3D spheroid culture system of ULM cells established in this study would be useful as a screening system for therapeutic agents. Trial registration number non-clinical trials

MiR-139-5p Regulates Proliferation, Apoptosis, and Cell Cycle of Uterine Leiomyoma Cells by Targeting TPD52 [Retraction

[This retracts the article DOI: 10.2147/OTT.S108890.].

Curcumol, a major terpenoid from Curcumae Rhizoma, attenuates human uterine leiomyoma cell development via the p38MAPK/NF-κB pathway.

Uterine fibroids (UFs) are the most common benign tumors in women of reproductive age. Curcumae Rhizoma, the main essential oil component of which is curcumol, is widely used for the treatment of phymatosis in China due to its antitumor, anti-inflammatory, antithrombin, anti-tissue fibrosis and anti-oxygen pharmacological activities, but its potential for the treatment of UFs has not been evaluated. This study aimed to investigate the effects and mechanisms of curcumol intervention in human uterine leiomyoma cells (UMCs). Putative targets of curcumol intervention in UFs were identified using network pharmacology strategies. Molecular docking was performed to assess the binding affinity of curcumol to core targets. A concentration gradient of curcumol (0, 50, 100, 200, 300, 400 and 500μM) or RU-486 (mifepristone, 0, 10, 20, 40, 50, and 100μM) was applied to UMCs, and cell viability was detected by the CCK-8 assay. Cell apoptosis and cell cycle were examined by flow cytometry, and cell migration was assessed by a wound-healing assay. Additionally, the mRNA and protein expression levels of critical pathway components were evaluated by RT‒PCR and western blotting. Finally, the actions of curcumol on different tumor cell lines were summarized. Network pharmacology predicted 62 genes with roles in the treatment of UFs with curcumol, and MAPK14 (p38MAPK) displayed a higher interaction degree. GO enrichment and KEGG analyses revealed that the core genes were abundantly enriched in the MAPK signaling pathway. The molecular binding of curcumol to core targets was relatively stable. In UMCs, 200, 300 and 400μM curcumol treatment for 24h decreased cell viability compared with that in the control group, and the greatest effect was detected at 48h and maintained until 72h. Curcumol arrested cells in the G0/G1 phase and subsequently suppressed mitosis, promoted early apoptosis and reduced the degree of wound healing in a concentration-dependent manner in UMCs. Furthermore, 200μM curcumol decreased the mRNA and protein expression of p38MAPK, the mRNA expression of NF-κB, and the protein expression of Ki-67 and increased the mRNA and protein expression of Caspase 9. Curcumol (300 and 400μM) decreased the mRNA and protein expression of p38MAPK, NF-κB, and Ki-67 and increased the protein expression of Caspase 9 in UMCs. Curcumol was demonstrated to treat tumor cell lines, including breast cancer, ovarian cancer, lung cancer, gastric cancer, liver cancer and nasopharyngeal carcinoma, but its effects on benign tumors have not yet been reported. Curcumol suppresses cell proliferation and cell migration while arresting the cell cycle in the G0/G1 phase and inducing cell apoptosis in UMCs via a mechanism related to p38MAPK/NF-κB pathway regulation. Curcumol may be a potential therapeutic and preventive agent in the treatment of benign tumors such as UFs.

Beta blockers reduce uterine fibroid incidence in hypertensive women

ObjectiveIs prior beta blocker (BB) use associated with reduced odds of the clinical incidence of leiomyomas? What is known alreadyIn-vitro and in-vivo evidence has supported the role of beta receptor blockade in reducing leiomyoma cell proliferation and growth. However, no population-based study to date has investigated this potential association. Study design, size, durationA nested case-control study was conducted in a population of women aged 18–65 with arterial hypertension (n = 699,966). Cases (n = 18,918) with a leiomyoma diagnosis were matched to controls (n = 681,048) with no such diagnosis at a 1:36 ratio by age and region of origin within the United States. Participants/materials, setting, methodsThis population was assembled from the Truven Health MarketScan® Research Database, which includes health insurance claims from January 1st, 2012 to December 31st, 2017. Prior use of BB wasdetermined fromoutpatient drug claims and leiomyoma development was indicated by a first-time diagnosis code. We conducted a conditional logistic regression to determine the odds of uterine fibroid development in women with prior use of BB compared to women with no such history. We then conducted subset analyses, stratifying the women by age group and by type of BB. ResultsWomen on a BB experienced 15% reduced odds of developing clinically recognized leiomyoma compared to non-users (OR 0.85, 95% CI 0.76–0.94). This association was significant for the 30–39 age group (OR 0.61, 95% CI 0.40–0.93) but no other age group. Of the BBs, propranolol (OR 0.58, 95% CI 0.36–95) demonstrated a significant association with reduced leiomyoma incidence and metoprolol (OR 0.82, 95% CI 0.70–0.97) was associated with lower uterine fibroid incidence after adjustment for comorbidities. ConclusionsHypertensive women with prior BB use experienced reduced odds of developing clinically recognized leiomyoma compared to non-users. A key predisposing risk factor for uterine leiomyoma is elevated blood pressure. Thus, the results of this analysis may have clinical relevance to women with hypertension, as the use of this drug may introduce a dual benefit of managing hypertension as well as curbing an increased risk of leiomyomas.

EZH2 activates Wnt/β-catenin signaling in human uterine fibroids, which is inhibited by the natural compound methyl jasmonate

To investigate the link between EZH2 and Wnt/β-catenin signaling and its role in uterine fibroids (UFs) pathogenesis and explore the potential effect of natural compound methyl jasmonate (MJ) against UFs. EZH2 overexpression or inhibition was achieved in human uterine leiomyoma (HuLM) cells using EZH2-expressing adenovirus or chemical EZH2 inhibitor (DZNep), respectively. The HuLM and normal uterine smooth muscle cells were treated with 0.1-3 mM of MJ, and several experiments were employed. Laboratory study. None. Methyl jasmonate. Protein expression of EZH2, β-catenin, and proliferating cell nuclear antigen (PCNA) was measured by Western blot as well as gene expression alterations of Wnt ligands (Wnt5A, Wnt5b, and Wnt9A), WISP1, CTNNB1, and its responsive gene PITX2 using quantitative real-time polymerase chain reaction. The protein and ribonucleic acid (RNA) levels of several markers were measured in MJ-treated or untreated HuLM cells, including EZH2 and β-catenin, extracellular matrix markers collagen type 1 (COL1A1) and fibronectin (FN), proliferation markers cyclin D1 (CCND1) and PCNA, tumor suppressor marker p21, and apoptotic markers (BAX, cytochrome c, and cleaved caspase 3). EZH2 overexpression significantly increased the gene expression of several Wnt ligands (PITX2, WISP1, WNT5A, WNT5B, and WNT9A), which increased nuclear translocation of β-catenin and PCNA and eventually HuLM cell proliferation. EZH2 inhibition blocked Wnt/β-catenin signaling activation where the aforementioned genes significantly decreased as well as PCNA, cyclin D1, and PITX2 protein expression compared with those in untreated HuLM. Methyl jasmonate showed a potent antiproliferative effect on HuLM cells in a dose- and time-dependent manner. Interestingly, the dose range (0.1-0.5 mM) showed a selective growth inhibitory effect on HuLM cells, not on normal uterine smooth muscle cells. Methyl jasmonate treatment at 0.5 mM for 24 hours significantly decreased both protein and RNA levels of EZH2, β-catenin, COL1A1, FN, CCND1, PCNA, WISP1, and PITX2 but increased the protein levels of p21, BAX, cytochrome, c and cleaved caspase 3 compared with untreated HuLM. Methyl jasmonate-treated cells exhibited down-regulation in the RNA expression of 36 genes, including CTNNB1, CCND1, Wnt5A, Wnt5B, and Wnt9A, and up-regulation in the expression of 34 genes, including Wnt antagonist genes WIF1, PRICKlE1, and DKK1 compared with control, confirming the quantitative real-time polymerase chain reaction results. Our studies provide a novel link between EZH2 and the Wnt/β-catenin signaling pathway in UFs. Targeting EZH2 with MJ interferes with the activation of wnt/β-catenin signaling in our model. Methyl jasmonate may offer a promising therapeutic option as a nonhormonal and cost-effective treatment against UFs with favorable clinical utility, pending proven safe and efficient in human clinical trials.

High levels of hypusinated eIF5A in leiomyoma and leiomyosarcoma pathologies: a possible novel therapeutic target

Research questionIs the hypusinated form of the eukaryotic translation initiation factor 5A (EIF5A) present in human myometrium, leiomyoma and leiomyosarcoma, and does it regulate cell proliferation and fibrosis? DesignThe hypusination status of eIF5A in myometrial and leiomyoma patient-matched tissues was evaluated by immunohistochemistry and Western blotting as well as in leiomyosarcoma tissues by immunohistochemistry. Myometrial, leiomyoma and leiomyosarcoma cell lines were treated with N1-guanyl-1,7-diaminoheptane (GC-7), responsible for the inhibition of the first step of eIF5A hypunization, and the proliferation rate was determined by MTT assay; fibronectin expression was analysed by Western blotting. Finally, expression of fibronectin in leiomyosarcoma tissues was detected by immunohistochemistry. ResultsThe hypusinated form of eIF5A was present in all tissues examined, with an increasing trend of hypusinated eIF5A levels from normal myometrium to neoplastic benign leiomyoma up to neoplastic malignant leiomyosarcoma. The higher levels in leiomyoma compared with myometrium were confirmed by Western blotting (P = 0.0046). The inhibition of eIF5A hypusination, with GC-7 treatment at 100 nM, reduced the cell proliferation in myometrium (P = 0.0429), leiomyoma (P = 0.0030) and leiomyosarcoma (P = 0.0044) cell lines and reduced the expression of fibronectin in leiomyoma (P = 0.0077) and leiomyosarcoma (P = 0.0280) cells. The immunohistochemical staining of leiomyosarcoma tissue revealed that fibronectin was highly expressed in the malignant aggressive (central) part of the leiomyosarcoma lesion, where hypusinated eIF5A was also highly represented. ConclusionsThese data support the hypothesis that eIF5A may be involved in the pathogenesis of myometrial benign and malignant pathologies.