Human Leiomyoma Cells Research Articles

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.

Adipocyte and Adipokines Promote a Uterine Leiomyoma Friendly Microenvironment.

Uterine leiomyomas are the most common benign tumors of the female reproductive system. Obese individuals have a higher burden of uterine leiomyoma, yet the mechanism relating obesity and leiomyoma development remains unknown. In this study, we observe the effect of adipocyte coculture and leptin treatment on human myometrium and leiomyoma cells. We isolated primary leiomyoma and myometrium cells from hysterectomy or myomectomy patients. Protein expression levels of phosphorylated ERK1/2/total ERK1/2, phosphorylated STAT3/total STAT3, and phosphorylated AKT1/2/3/total AKT1/2/3 were quantified using immunoblotting in immortalized and primary leiomyoma and myometrial cells cocultured with human adipocytes and treated with leptin. An enzyme-linked immunosorbent assay (ELISA) was used to assess pro-inflammatory, fibrotic, and angiogenic factors in immortalized human myometrium and leiomyoma cells treated with leptin. The effects of STAT3, ERK, and AKT inhibitors were assessed in leiomyoma cell lines additionally cultured with adipocytes. Adipocyte coculture and leptin treatment increases the expression of JAK2/STAT3, MAPK/ERK, and PI3K/AKT signaling while inhibitors suppressed this effect. Leptin induces a tumor-friendly microenvironment through upregulation of pro-inflammatory (IFNγ, IL-8, IL-6, GM-CSF, MCP-1, and TNF-α), fibrotic (TGF-β1, TGF-β2, and TGF-β3), and angiogenic (VEGF-A, HGF, and Follistatin) factors in human leiomyoma cells. Furthermore, adipocyte coculture and leptin treatment increases leiomyoma cells growth through activation of MAPK/ERK, JAK2/STAT3, and PI3k/AKT signaling pathways. Finally, STAT3, ERK, and AKT inhibitor treatment suppressed PCNA, TNF-α, TGF-β3, and VEGF-A intracellular staining intensity in both adipocyte coculture and leptin treated leiomyoma cells. These findings suggest that, in obese women, adipocyte secreted hormone or adipocytes may contribute to leiomyoma development and growth by activating leptin receptor signaling pathways.

Pivotal Role of Ubiquitin Carboxyl-Terminal Hydrolase L1 (UCHL1) in Uterine Leiomyoma.

Uterine leiomyomas are smooth-muscle tumors originating in the myometrium and are the most common pelvic tumors in women of reproductive age. Symptomatic tumors may result in abnormal uterine bleeding, bladder dysfunction, pelvic discomfort, and reproductive issues, such as infertility and miscarriage. There are currently few non-invasive treatments for leiomyoma, but there are no practical early intervention or preventive methods. In this study, human uterine leiomyoma and myometrial tissues were used to detect the protein and mRNA expression levels of UCHL1. To explore the effects of UCHL1 knockdown and inhibition in leiomyoma and myometrial cells, we determined the mRNA expressions of COL1A1 and COL3A1. Collagen gel contraction and wound-healing assays were performed on myometrial and leiomyoma cells. We found that UCHL1 expression was considerably higher in uterine leiomyomas than in the myometrium. COL1A1 and COL3A1 expression levels were downregulated after inhibition of UCHL1 in human leiomyoma cells. Furthermore, the elimination of UCHL1 significantly decreased the migration and contractility of leiomyoma cells. In conclusion, these results indicate that UCHL1 is involved in the growth of leiomyoma in humans. For the treatment of uterine leiomyoma, targeting UCHL1 activity may be a unique and possible therapeutic strategy.

Leptin induces leiomyoma cell proliferation and extracellular matrix deposition via JAK2/STAT3 and MAPK/ERK pathways

To investigate the molecular effects of leptin on uterine leiomyoma cells. Experimental study using invitro culture of immortalized human leiomyoma (HuLM) cells. Academic university center. Women with uterine fibroids who underwent a hysterectomy or myomectomy. Administration of human recombinant leptin to the media of cultured HuLM cells separately or in combination with pharmacologic Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) or mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) inhibitors. We examined HuLM tissues and cells for the expression of the leptin receptor, termed OB-R. Cellular proliferation was measured at 6, 24, and 48 hours using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. Protein expression levels of proliferating cell nuclear antigen, collagen 1, phosphorylated STAT3/total STAT3, and phosphorylated ERK1/2 and total ERK1/2 were quantified using immunoblotting. Pharmacologic inhibitors were employed to further assess the role of the JAK2/STAT3 and MAPK/ERK pathways in the proliferative response. The presence of OB-R was confirmed in clinical leiomyoma and myometrial tissue obtained from 3 separate human subjects using immunofluorescence staining, and the expression of OB-R in HuLM cells was identified using immunoblotting. There was no significant difference in the expression of the leptin receptor in the myometrium compared with that in the leiomyoma tissue. Leptin stimulated cell proliferation and extracellular matrix (ECM) deposition at 24 hours after treatment. Pretreatment with a JAK2/STAT3 inhibitor attenuated ECM deposition, and pretreatment with a MAPK/ERK inhibitor significantly decreased leptin's stimulatory effect on cell proliferation and ECM deposition. Leptin induces a proliferative response and ECM deposition in HuLM cells. These findings suggest that leptin, acting through the JAK2/STAT3 and MAPK/ERK pathways, is involved in the development of uterine leiomyomas, which may partly explain their increased incidence in obese women.

Novel effects of piperlogumine on uterine fibroid tumor: An in vitro mouse model study

Purpose: To investigate the in vivo anti-tumor effect of piperlongumine (PL) in a mouse model of leiomyoma xenograft and in leiomyoma cell lines.&#x0D; Methods: The anti-proliferative effect of PL on ELT-3 cells was determined using MTT assay. Human and rat leiomyoma cells were used for the in vitro investigations. Rat leiomyoma cell lines were treated with various PL concentrations (50 - 100 μM)) for 48 h. Immunodeficient mice were subcutaneously injected with varying doses of estrogen or progesterone, and xenografted with explanting human leiomyoma cells in in vivo experiments. Proliferation assessment, caspase-3 expression, analysis of tumour samples, insertion of pellets of oestrogen-progesterone, tissue treatment and implantation, and immuno-histochemical analyses were carried out using appropriate procedures.&#x0D; Results: Piperlongumine (PL) produced significant and dose-dependent increase in caspase-3 activity, apoptosis and suppression of cellular proliferation (p &lt; 0.01). Moreover, Western blot data demonstrated that PL decreased phosphorylation of Akt signaling pathway. The results showed significant (p &lt; 0.01) inhibition of tumor growth, including in ultra-sound in vivo studies, when compared with 30-day control and animals treated with PL (100 μg/g). Immuno-histochemical studies showed that PL decreased the expression of proliferation marker in xenografted tumor tissues (p &lt; 0.02).&#x0D; Conclusion: These results suggest that piperlongumine has potentials as a therapeutic agent for the management of uterine leiomyoma. However, additional studies using human cell lines are required to understand its genetic and molecular mechanisms.

Hypoxia Induces Proliferation via NOX4-Mediated Oxidative Stress and TGF-β3 Signaling in Uterine Leiomyoma Cells

ABSTRACT Uterine leiomyomas, the most common tumors of the female reproductive system, are known to have a hypoxic microenvironment. However, the role of such environment in leiomyoma pathobiology remains unknown. The objective was to determine the effects of hypoxia on leiomyoma cells, and the mechanisms. We found that hypoxia induces proliferation and inhibits apoptosis in human leiomyoma cells. This pro-proliferative effect was accompanied by an increase in reactive oxygen species (ROS) generation and the expression of NADPH oxidase 4 (NOX4). The specific NOX4 inhibitor GLX351322 abrogated this hypoxia-induced ROS generation, cellular proliferation, and apoptosis inhibition. To further investigate the mechanism of NOX4-mediated proliferation, we treated leiomyoma cells grown in normoxia with media from leiomyoma cells cultured under hypoxia. This resulted in increased ROS generation and NOX4 expression, suggesting the hypoxia-induced effects are mediated by an autocrine mechanism. We worked to identify the nature of this autocrine factor. We found that the expression of TGF-β3 and its downstream signaling target pSmad3, are increased in hypoxic leiomyoma cells. To examine the hypothesis that TGF-β3 is, at least, a part of this autocrine mechanism, we treated hypoxic leiomyoma cells with the HIF-1α inhibitor KC7F2 which we discovered to ameliorate the hypoxia-induced TGF-β3 expression. Furthermore, pharmacologic inhibition with the TGF-β/Smad inhibitor SB431542 reduced hypoxia-induced NOX4 expression and ROS generation and attenuated cell proliferation. Thus, we have identified a novel mechanism by which hypoxia induces proliferation in leiomyoma cells. This finding adds to our understanding of leiomyoma pathobiology and can help in identifying new therapeutic targets. Supplemental data for this article can be accessed here .

Simvastatin Inhibits Wnt/β-Catenin Pathway in Uterine Leiomyoma.

The Wnt/β-catenin pathway is upregulated in uterine leiomyomas, the most common benign tumors in the female reproductive tract. Simvastatin is an antihyperlipidemic drug, and previous in vitro and in vivo reports showed that it may have therapeutic effects in treating leiomyomas. The objective of this study was to examine the effects of simvastatin on the Wnt/β-catenin signaling pathway in leiomyoma. We treated primary and immortalized human leiomyoma cells with simvastatin and examined its effects using quantitative real-time polymerase chain reaction, Western blotting, and immunocytochemistry. We also examined the effects using human leiomyoma tissues from an ongoing randomized controlled trial in which women with symptomatic leiomyoma received simvastatin (40 mg) or placebo for 3 months prior to their surgery. The results of this study revealed that simvastatin significantly reduced the expression of Wnt4 and its co-receptor LRP5. After simvastatin treatment, levels of total β-catenin and its active form, nonphosphorylated β-catenin, were reduced in both cell types. Additionally, simvastatin reduced the expression of Wnt4 and total β-catenin, as well as nonphosphorylated β-catenin protein expression in response to estrogen and progesterone. Simvastatin also inhibited the expression of c-Myc, a downstream target of the Wnt/β-catenin pathway. The effect of simvastatin on nonphosphorylated-β-catenin, the key regulator of the Wnt/β-catenin pathway, was recapitulated in human leiomyoma tissue. These results suggest that simvastatin may have a beneficial effect on uterine leiomyoma through suppressing the overactive Wnt/β-catenin pathway.

Hop and artichoke extracts inhibit expression of extracellular matrix components in uterine leiomyoma cells

To screen 14 different plant extracts for their antifibrotic effect on human primary leiomyoma and healthy myometrial cells. Preclinical study. University research laboratory. Human uterine leiomyoma and matched myometrial tissues were obtained from Caucasian premenopausal women with symptomatic uterine fibroids at the time of hysterectomy. Primary human leiomyoma and myometrial cells were cultured in the absence or presence of the plant extracts. Quantification of the expression of extracellular matrix components, such as fibronectin 1 (FN1), collagen type I alpha 1 (COL1A1), and versican (VCAN), and the profibrotic growth factor activin A or inhibin beta A subunit (INHBA). The cells were treated with the 14 extracts for 48 hours, and we measured FN1 messenger RNA (mRNA) expression. Of the 14 extracts, about (ABO) ABO-2 (hop) and ABO-9 (artichoke) significantly reduced FN1 expression in both the cell types. Next, we evaluated the effect of fractions of these 2 extracts on the mRNA expression of FN1 and other extracellular matrix components, such as COL1A1, VCAN, and INHBA, in leiomyoma and myometrial cells. We found that ABO-2 (hop) and ABO-9 (artichoke) as well as their fractions, ABO-AR-2016-015 (fraction of ABO-2) and ABO-AR-2014-168 (fraction of ABO-9), reduced the mRNA expression of FN1, COL1A1, VCAN, and INHBA in primary leiomyoma cells. In primary myometrial cells, the mRNA expression of FN1, COL1A1, VCAN, and INHBA was not greatly affected. These results suggest that the hop and artichoke extracts possess antifibrotic properties and support additional evaluation using invivo models.

EVALUATING THE INHIBITORY EFFECT OF ELAGOLIX & RELUGOLIX ON LEIOMYOMA GROWTH IN 2D CELL CULTURE

Elagolix and relugolix, both oral GnRH antagonists, are structurally similar; however their pharmakinetic properties differ. At the GnRH receptor elagolix’s IC50 is 1.5 nM with a half-life of 4-6 hours, while relugolix’s IC50 is 0.33 nM with a half-life of 49 hours. The goal of this study is to compare the effect elagolix and relugolix have on the expression of extracellular matrix (ECM) proteins in human leiomyoma cells in 2D cell culture. We hypothesized that both GnRH antagonists directly impact ECM protein expression relative to untreated leiomyoma cells. Leiomyoma cells were grown 2-dimentionally and exposed to elagolix and relugolix for 24 and 48 hours. Expression of various ECM proteins and GnRHR were analyzed using western blot and immunohistochemistry. At 100 nM relugolix (n=10) resulted in maximal, decreased expression of col1A1 at 24 hours (1.78 ± 0.06-fold; p<0.01) and 48 hours (1.92 ± 0.14-fold; p<0.04) and the difference between the two time points was not significant (p=0.87). Elagolix (n=11) resulted in maximal, decreased expression of col1A1 at 24 hours (1.56 ± 0.14-fold; p<0.03) but suppression was lost by 48 hours; the difference between the time points was significant (p<0.01). At 100 nM relugolix resulted in maximal, decreased expression of fibronectin at 24 hours (1.7 ± 0.07-fold; p<0.01) and 48 hours (1.8 ± 0.07-fold; p<0.01) with the difference between the two time points not being significant (p=0.52); elagolix resulted in maximal, decreased expression of fibronectin at 24 hours (1.7 ± 0.14-fold; p=0.01) and 48 hours (2.0 ± 0.09-fold; p<0.01) with no significant difference between the time points (p=0.19). An upregulation was seen in versican expression when cells were treated with relugolix for 24 hours though no concentration achieved growth to statistical significance when compared to untreated cells. A significant, concentration dependent downregulation was achieved at 48 hours (p<0.01) and at 100 nM relugolix resulted in maximal, decreased expression of versican (1.5 ± 0.07-fold; p<0.01). At 24 hours maximal, decreased expression of versican was seen at 100 nM (1.6 ± 0.10-fold; p<0.01) for cells treated with elagolix; a statistically significant upregulation was seen at 48 hours (p=0.02). In terms of the GnRH receptor, no significant difference between GnRH receptor protein expression in relugolix and elagolix was seen at 24 hours (p=0.57). At 48 hours, GnRH receptor protein expression increased in cells treated with elagolix when compared to cells treated with relugolix (p=0.037) suggesting the suppressive effect of elagolix at the receptor is lost by 48 hours. Our findings suggest that treatment with relugolix and elagolix directly regulated collagen, fibronectin, versican, and GnRH receptor protein expression in our 2D human leiomyoma cell culture model.

MicroRNA-139-5p Regulates Fibrotic Potentials via Modulation of Collagen Type 1 and Phosphorylated p38 MAPK in Uterine Leiomyoma.

PurposeThis study aimed to elucidate whether microRNA-139-5p is involved in the pathogenesis of uterine leiomyoma.Materials and MethodsHuman leiomyoma and matched human smooth muscle samples were obtained from 10 women who underwent hysterectomy for uterine leiomyoma. MicroRNA (miRNA) expression was analyzed by quantitative real-time polymerase chain reaction. To assess the effects of miR-139-5p on cultured leiomyoma cells, cell migration, collagen gel contraction, wound healing, and the expression levels of hallmark proteins were evaluated in cells transfected with a miR-139-5p mimic.ResultsThe expression of miR-139-5p was significantly lower in leiomyoma tissues than in matched smooth muscle tissues. Restored miR-139-5p expression in miR-139-5p mimic-transfected human leiomyoma cells resulted in decreased contractility of the ECM and cell migration. In addition, upregulation of miR-139-5p decreased the protein expression of collagen type 1 and phosphorylated p38 MAPK.ConclusionExpression of miR-139-5p is downregulated in leiomyoma cells and modulation of miR-139-5p may be involved inthe pathogenesis of leiomyomas through the regulation of collagen type 1 and phosphorylated p38 MAPK. Therefore, miR-139-5p is a potential therapeutic target for leiomyoma.

HIF1-ALPHA INDUCED ADRENOMEDULLIN EXPRESSION SUPPORTS ANGIOGENESIS IN 3D LEIOMYOMA XENOGRAFTS

Cellular responses to hypoxia are critical to homeostasis. In eukaryotes, the primary regulator of angiogenesis is the transcription factor hypoxia inducible factor-1 alpha (HIF-1α). Hormonally-stimulated human leiomyoma 3D xenografts express HIF1-α protein and undergo in vivo neovascularization. Our objective was to characterize hypoxia regulated genes and HIF-1α regulated angiogenesis in our human leiomyoma cell lines and xenograft model.

Activin A induces tumorigenesis of leiomyoma via regulation of p38β MAPK-mediated signal cascade

ObjectiveThe objective of this study was to investigate the role of p38-C/EBPβ signaling in leiomyoma cells and myometrial cells challenged with Activin A, and to identify specifically the isoform of p38 MAPK that mediates the effects of Activin A. MethodsThe immortalization human leiomyoma cells (HuLM) and human myometrial cells (HM), and mouse myometrial tissues were treated with Activin A (4 nM) in response to p38α/β inhibition (10 μM SB202190) or depletion (p38 α/β-targeting siRNA or p38β muscle specific-knock out mice). p38 MAPK signaling molecules (p-p38 and p-C/EBPβ) and ECM components (COL1A1 and/or FN) were analyzed by Western blotting. ResultsActivin A induced ECM accumulation in leiomyoma cells and myofibroblastic transformation in myometrical cells specifically by p38β MAPK. ConclusionThis study is the first to demonstrate that activation of C/EBPβ by p38β MAPK may contribute to tumorigenesis and progression of Activin A-induced leiomyoma. Specific p38β inhibition may represent a novel and promising intervention for leiomyoma.

Pigment epithelium derived factor as a novel multi-target treatment for uterine fibroids.

Does recombinant pigment epithelium derived factor (PEDF) have potential in treating uterine fibroids? In-vitro models that used human leiomyoma and Eker rat uterine leiomyoma (ELT-3) cell lines. The ELT-3 cell line was used to examine cellular targets after adding recombinant PEDF to the culture media. Athymic nude female mice were used as an in-vivo model. They were injected with ELT-3 cells to induce ectopic fibroid lesions, then treated with recombinant PEDF. RNA expression of PEDF and its receptors was found in both leiomyoma cell lines, as well as the expression of PEDF receptors. Addition of recombinant PEDF to the culture medium of leiomyoma cell lines activated ERK in a time-dependent manner, induced down-regulation of vascular endothelial growth factormRNA and protein, as well as the mRNAs of oestrogen receptors alpha and beta and inhibited cellular proliferation. Treatment of mice-bearing fibroids with recombinant PEDF reduced fibroid growth rate and resulted in smaller tumours. This study suggests that recombinant PEDF is a putative novel potent physiological treatment for uterine fibroids. It targets several cornerstones of fibroid pathobiology in parallel, including vascular endothelial growth factor and oestrogen receptors, which are needed for vascularization, and restricts fibroid growth and final size in an animal model.

Steroid hormones and hormone antagonists regulate the neural marker neurotrimin in uterine leiomyoma

To characterize the role of steroid hormone and antihormone exposure on neurotrimin (NTM) expression in human leiomyoma and myometrial tissue and cells. Laboratory study of placebo and ulipristal acetate (UPA)-treated patient tissue. Invitro assessment of immortalized myometrial and leiomyoma cell lines after hormone and antihormone exposure. Academic research center. Not applicable. Exposure of leiomyoma cell lines to 17β-E2, medroxyprogesterone acetate (MPA), UPA, and fulvestrant. Messenger RNA expression quantified with the use of RNASeq analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Protein levels quantified by means of Western blot analysis. Immunohistochemistry (IHC) on placebo- and UPA-treated patient uterine tissue specimens. Expression of NTM in human uterine leiomyoma specimens according to RNASeq was increased compared with myometrium (5.22 ± 0.57-fold), which was confirmed with the use of qRT-PCR (1.95 ± 0.05). Furthermore, NTM protein was elevated in leiomyoma tissue compared with matched myometrium (2.799 ± 0.575). IHC revealed increased staining intensity in leiomyoma surgical specimens compared with matched myometrium of placebo patients. Western blot analysis in immortalized leiomyoma cell lines demonstrated an up-regulation of NTM protein expression (2.4 ± 0.04). Treatment of leiomyoma cell lines with 17β-E2 yielded a 1.98 ± 0.11-fold increase in NTM protein expression; however, treatment with fulvestrant showed no significant change compared with control. Leiomyoma cell lines demonstrated a 1.91 ± 0.97-fold increase in NTM protein expression after progesterone treatment. RNASeq analysis demonstrated a reduced expression in patient leiomyoma after UPA treatment (0.75 ± 0.14). Treatment of leiomyoma cells with UPA demonstrated a reduced total NTM protein amount (0.54 ± 0.31) in patients, which was confirmed with the use of IHC (UPA10 147.2 ± 9.40, UPA20 182.8 ± 8.98). Invitro studies with UPA treatment revealed a concentration-dependent effect that supported these findings. NTM, a neural cell adhesion molecule, is increased in leiomyoma compared with myometrium in patient tissue and invitro models after estrogen and progesterone treatment. Down-regulation of expression occurs after UPA treatment, but not after fulvestrant exposure. NCT00290251.

CORRIGENDUM FOR "Role of Activin-A and Myostatin and Their Signaling Pathway in Human Myometrial and Leiomyoma Cell Function".

CORRIGENDUM FOR "Role of Activin-A and Myostatin and Their Signaling Pathway in Human Myometrial and Leiomyoma Cell Function".

Simvastatin inhibits RhoA activation, collagen expression and gel contraction in human leiomyoma cells

Simvastatin inhibits RhoA activation, collagen expression and gel contraction in human leiomyoma cells

Effect of simvastatin on integrin-β1 and its downstream mediators in human leiomyoma cells

Effect of simvastatin on integrin-β1 and its downstream mediators in human leiomyoma cells

CANCER ASSOCIATED fiBROBLASTS (CAFS) INflUENCE TISSUE INVASION ON SALIVARY GLAND MUCOEPIDERMOID CARCINOMA (MEC) CELLS

Objectives MEC is the most common salivary gland malignancy. Although prognosis is mostly based on TNM status, histologic grade is also used as a parameter to determine treatment. CAFs have been reported to influence worse behavior in several malignancies including head and neck squamous cell carcinoma. We noticed the presence of CAF-like cells, displaying immunohistochemical positivity for alpha smooth muscle actin, in some MECs with bad outcome and we hypothesize that CAFs may influence MEC aggressiveness. Therefore, we investigated tissue invasion using the organotypic 3D human leiomyoma model and cell migration using the Incucyte® system with a gel derived from human leiomyomas (myogel). MEC cell lines HMC2 and UTMUC1, derived from high grade tumors, were cultivated alone or co-cultured with CAFs in order to evaluate if CAFs would influence MEC cells invasion and migration. Cells were cultivated on top of human leiomyoma discs for 14 days to allow invasion. Discs were fixed in 10% buffered formalin, processed and 3 micrometer tissue slices were prepared and submitted to immunohistochemical reaction with a pan-cytokeratin antibody (clone AE1/AE3). The number of invasive cells was determined by counting invasive cells under light microscope. Invasion was studied using a wound scratch assay coupled with a live camera and data obtained was analyzed using software provided by the manufacturer. Findings Both MEC cell lines (HMC2 and UTMUC1) displayed a significant increase in tissue invasion when co-cultured with CAFs compared to when they were cultured alone. Only HMC2 cell line presented a significant increase in migration when co-cultured with CAFs. Conclusion CAFs significantly increase MEC cell lines invasion and migration. The presence of CAFs deserves further investigation in MEC tumor samples and it may correlate with tumor behavior and clinical outcome.

A Culture System Similar to the Embryonal Microenvironment Supports Transdifferentiation in Human Leiomyoma Cells

Current research on stem cells and regenerative medicine indicates new perspectives on the relationship between differentiation and gene information. Induced pluripotent stem (iPS) cells need the artificial gene expression of the somatic cell, which is related to initialization. Paradoxically, that means that cell differentiation depends on almost all the gene information stored precisely in the nucleus of a somatic cell, plus the transformation of gene expression. Our research team tried to identify the culture conditions in the transdifferentiation of human leiomyoma cells, closely similar to the early embryonal stage, composed of various factors (hypoxia, non-serum, and regulation of cell adhesion molecules such as Wnt/β-catenin signaling). As a result, inhibition of Wnt/β-catenin signaling under serum starvation and hypoxia induces adipocytic transdifferentiation in human leiomyoma cells. Here we explain this unique culture system, referring to the components of intracellular mechanisms and the extracellular microenvironment in embryo development.

Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) Hull Extract and Active Compounds Inhibit Proliferation of Primary Human Leiomyoma Cells and Protect against Sexual Hormone-Induced Mice Smooth Muscle Hyperproliferation.

Uterine leiomyomas, also known as fibroids, are benign neoplasms of the uterus and have a high incidence rate in women of reproductive age. Hysterectomy or myomectomy is the initial treatment, but fibroids will recur if the patient is still exposed to similar risk factors. Therefore, developing new therapeutic strategies are urgently necessary. In this study, the anti-proliferation effects of each fraction of adlay seeds were evaluated in uterine leiomyomas, and we identified the potential phytochemical compounds. We found that the ethyl acetate fraction of adlay hull (AHE-ea) appeared to be highly efficient in the anti-proliferation of rat uterine leiomyoma ELT3 cells and primary human uterine leiomyoma (hUL) cells. The proliferation of primary human normal uterine smooth muscle (UtSMC) and normal uterine myometrial (hUM) cells were also suppressed by AHE-ea. Two phytosterols, stigmasterol and β-sitosterol, were identified from AHE-ea fraction. Mice treated with AHE-ea and stigmasterol alone demonstrated reduced diethylstilbestrol/medroxyprogesterone 17-acetate (DES/MPA)-induced uterine myometrial hyperplasia, which is the critical step for the development of leiomyoma. Taken together, our results suggest that the AHE-ea fraction could be considered as a natural plant-based medicine in the prevention or treatment of uterine leiomyoma growth.