Leiomyoma Cell Lines Research Articles

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.

NAV3, a Tumor Suppressor Gene, Is Decreased in Uterine Leiomyoma Tissue and Cells.

NAV 3 is a tumor suppressor of unknown function in leiomyomas. The objective of this study is to assess NAV3 expression and its potential role in human uterine leiomyomas. NAV3 protein expression was examined in patient leiomyoma and patient-matched myometrial tissue samples by Western blot and immunohistochemistry. NAV3 mRNA and protein expression was assessed in leuprolide acetate- and cetrorelix-treated cell line leiomyoma samples. RNAseq analysis of placebo-treated leiomyoma compared with myometrium demonstrated the presence of transcripts encoding for several neuronal proteins. For NAV3, RNA sequence analysis demonstrated decreased expression in leiomyoma as compared with myometrium (0.86 ± 0.03 fold). Presence of NAV3 mRNA was also decreased in leiomyoma surgical samples (0.43 fold ± 0.05, p = 0.026) compared with patient-matched myometrium. Confirmatory qRT-PCR results on immortalized leiomyoma and myometrial cell lines similarly demonstrated a decrease in expression of NAV3 in leiomyomas (0.28 ± 0.02, p = 0.00075). Immunohistochemical analysis demonstrated a significant decrease in NAV 3 protein in leiomyomas (H-score 154.7 ± 6.2) as compared with myometrium (H-score; 312.5 ± 14.7, p < 0.0001). Leuprolide acetate-treated leiomyoma cells demonstrated an increase in NAV 3 mRNA expression (1.53 ± 0.13, p < 0.0001). Similarly, Western blot analysis on leuprolide-treated leiomyoma cells showed a non-significant increase in NAV 3 protein expression (1.26 ± 0.09, p = 0.063). NAV 3, a tumor suppressor in numerous cancers, is decreased in leiomyoma cells and tissue compared with myometrium, and increased by GnRH analog treatment, suggesting that NAV3 may mediate steroid hormone-independent leiomyoma regulation by GnRH analogs.

Isoliquiritigenin induces cell death programs and reduces ECM accumulation in uterine leiomyoma

Abstract Background Uterine leiomyomas, also known as fibroids, are the comment and high prevalence in women of reproductive age. Isoliquiritigenin (ISL), a licorice flavonoid, exerts variant biological properties. In this study, the effect of ISL on anti-proliferation of uterine leiomyoma was investigated. Methods Cell viability was determined using the MTT assay. The protein expression was assayed using Western blot analysis. Cell apoptosis was assayed using Annexin V-FITC/PI and Hoechst 33342 staining. Results The results showed that treatment of uterine leiomyoma ELT3 cells and primary uterine smooth muscle UtSMC cells with ISL alone or ISL plus estradiol (E2) reduced cell proliferation. Cotreatment with E2 and ISL inhibit G2/M and S phase arrest in ELT3 and UtSMC cells, respectively. The higher sub-G1 phase arrest and fluorescence intensity of Hoechst 33342 nucleus stain were observed in cotreatmenet of ELT3 cells with E2 and ISL compared with UtSMC cells. Furthermore, cotreated ELT3 cells with E2 and ISL induced apoptosis and autophagy cell death programs. The E2-enhanced mice uterine myometrium growth was reduced by ISL presence, resulting in reduced the myometrium layer. Moreover, ISL reduced extracellular matrix proteins and matrix metalloproteinase (MMPs) expression, whereas increased tissue inhibitor of MMPs (TIMPs) expression. Conclusion Our findings indicated the inhibitory effects of ISL on the animal model and indicated that ISL reduced ECM-related protein expression and cell proliferation in the leiomyoma cell line. Taken together, our results suggested that ISL could be considered as a new option for the treatment of uterine leiomyoma.

IL6 and STAT-3 pathway highlight the differences in molecular responses in myometrium and uterine fibroids

Human fibroids are highly prevalent and symptomatic uterine tumors. Phenotypically, they are different from normal myometrium because of massive production of extracellular matrix (ECM), which is a hallmark of these benign tumors resulting in symptoms including abnormal bleeding and pain. Dysregulated production of inflammatory cytokine, IL6 and its regulated JAK/STAT-3 pathway are known to contribute to the fibrotic process. Our objective was to understand the effect of IL6 on fibroid and myometrium cells and the role of JAK/STAT pathway in production of matrix proteins that play a major role in fibroid pathogenesis. Laboratory study. Leiomyoma and patient-matched myometrium cell lines exposed to different concentrations of IL6 and JAK/STAT-3 modulators for various exposure time periods, in a 2D culture model system. Changes in expression of ECM proteins and regulating pathways were assessed using western blot. In leiomyoma cells, IL6 activation of STAT-3 peaked (2.32+/- 0.21 fold) as early as 1.5hr of continuous exposure, and was 1.4+/- 0.04 fold increased at end of 3hr of exposure. The maximum increase (1.59+/- 0.07 fold) in the feedback loop protein, Suppressor of Cytokine Signaling (SOCS3) required a short exposure to IL6 (30min) followed by collection after 3hr. We also observed an increase (1.9+/- 0.11 fold) in the ECM structural collagen-1 after continuous exposure to IL6 (3hr) in leiomyoma cells. No significant effect of IL6 was observed in myometrium cells. Collagen-1 protein was also affected significantly in the leiomyoma cells on use of JAK/STAT-3 modulators. An increase of 2.21+/-0.052 fold was observed on direct activation of the pathway in leiomyoma cells. Increase were also observed in ECM protein fibronectin in response to the JAK/STAT-3 modulators. Response of transforming growth factor beta3 (TGFβ3) protein, involved in fibrotic specific pathway, on use of JAK/STAT-3 modulators indicated that both pathways may interact to regulate the production of the ECM proteins in leiomyomas. Direct effect of inflammatory cytokine IL6, on leiomyoma and not myometrium cells indicate that this differential response to IL6 and JAK/STAT3 pathway may be the key to increased ECM production and growth in uterine leiomyomas.

Abstract 3022: Dissecting rapamycin resistance in a Tsc2-null rat leiomyoma cell line developed in a murine xenograft

Abstract Despite the identification of mTOR hyperactivation as the main biochemical defect downstream of TSC1/TSC2 inactivation in Lymphangioleiomyomatosis (LAM) and Tuberous Sclerosis Complex (TSC), and the approval of rapamycin and rapalogs for the treatment of the related lesions, these drugs are cytostatic and continued treatment is required for clinical benefit. The latter raises the possibility of acquired drug resistance over long-term use. To explore the mechanisms leading to rapamycin resistance in LAM/TSC, we xenografted SCID mice with ELT3 cells (Tsc2-null derived from an Eker rat uterine leiomyoma), explanted a tumor that was not responsive to rapamycin, and generated cell line ELT3-245. The cell growth of ELT3-245 is not inhibited by rapamycin in vitro. SCID mice inoculated with ELT3-245 form palpable tumors significantly faster, compared to ELT3 cells, respond very poorly to rapamycin at the initial stages of treatment, but soon become non-responsive. In addition, these cells form macroscopically visible metastases to the lungs of ELT3-245 tumor-bearing mice. We performed global gene expression profiling, comparing ELT3-245 to ELT3 cells, and found that the rapamycin-resistant cells have decreased expression of genes associated with epithelial cells and increased expression of genes associated with mesenchymal-like characteristics. Relative gene expression of Myc, Egfr, Akt2, Jun, and Ocln were assessed by RT-qPCR in ELT3 vs ELT3-245 after 24h of treatment with 20 nM rapamycin or DMSO. Important discrepancies include a 2.8-fold increased expression of Egfr under rapamycin treatment in ELT3 cells but not in ELT3-245; however, ELT3-245 had a 15-fold higher expression of Egfr prior to treatment, compared to ELT3. In addition, ELT3-245 cells strikingly increase expression of Myc when challenged with rapamycin, compared to ELT3. Similarly, Akt2 is increased considerably under rapamycin treatment in ELT3-245 and is already higher prior to treatment compared to ELT3. Finally, Ocln expression was at least 100-fold reduced in ELT3-245, compared to ELT3, both under rapamycin treatment and no-treatment conditions. These results suggest that Myc, Egfr, Akt2, and Ocln are among the genes highly relevant to acquired rapamycin resistance in this Tsc2-null rat leiomyoma cell line and point toward the pathways that need to be interfered with in order to overcome it. Citation Format: Natalia Filippidou, Mathildi Valianou, Daniel L. Johnson, John J. Bissler, Aristotelis Astrinidis. Dissecting rapamycin resistance in a Tsc2-null rat leiomyoma cell line developed in a murine xenograft [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3022.

Ulipristal acetate decreases active TGF-β3 and its canonical signaling in uterine leiomyoma via two novel mechanisms

To characterize the effect of ulipristal acetate (UPA) treatment on transforming growth factor (TGF) canonical and noncanonical signaling pathways in uterine leiomyoma tissue and cells. UPA decreased extracellular matrix in surgical specimens; we characterize the mechanism in this study. Laboratory study. University. Exposure of leiomyoma cell lines to UPA. RNAseq was performed on matched myometrium and leiomyoma surgical specimens of placebo- and UPA-treated patients. Changes in gene expression and protein were measured using quantitative polymerase chain reaction and western immunoblot analysis, respectively. In surgical specimen, mRNA for TGF-β3 was elevated 3.75-fold and TGFR2 was decreased 0.50-fold in placebo leiomyomas compared with myometrium. Analysis of leiomyomas from UPA-treated women by western blot revealed significant reductions of active TGF-β3 (0.64 ± 0.12-fold), p-TGFR2 (0.56 ± 0.23-fold), pSmad 2 (0.54 ± 0.04-fold), and pSmad 3 (0.65 ± 0.09-fold) compared with untreated leiomyomas. UPA treatment demonstrated statistically significant reduction in collagen 1, fibronectin, and versican proteins. Notably, there was a statistically significant increase of the extracellular matrix protein fibrillin in leiomyoma treated with UPA (1.48 ± 0.41-fold). Data from invitro assays with physiologic concentrations of UPA supported the invivo findings. TGF-β pathway is highly up-regulated in leiomyoma and is directly responsible for development of the fibrotic phenotype. UPA attenuates this pathway by reducing TGF-β3 message and protein expression, resulting in a reduction in TGF-β canonical signaling. In addition, UPA significantly increased fibrillin protein expression, which can serve to bind inactive TGF-β complexes. Therefore, UPA inhibits leiomyoma fibrosis by decreasing active TGF-β3 and diminishing signaling through the canonical pathway. NCT00290251.

Berberine Inhibits Uterine Leiomyoma Cell Proliferation via Downregulation of Cyclooxygenase 2 and Pituitary Tumor-Transforming Gene 1.

We previously demonstrated that berberine (BBR) inhibits cell proliferation and induces apoptosis in a human uterine leiomyoma (UtLM) cell line but does not demonstrate a significant cytotoxic effect in a normal human uterine smooth muscle (UtSM) cell line. However, the mechanisms of this inhibition are unclear. Of note, cyclooxygenase 2 (COX2) and pituitary tumor-transforming gene 1 (PTTG1) are overexpressed in human uterine leiomyomata and are involved in the pathogenesis of uterine fibroids (UFs). We found that COX2 and PTTG1 were overexpressed in UtLM and that BBR decreased COX2 and PTTG1 expression in UtLM cells. Our data support that UtLM and UtSM are immortalized cell lines without phenotypic alterations from parental cell types and suggest that COX2 and PTTG1 are molecular targets for BBR. However, studies in these cell lines may not reveal all activities of BBR in vivo, and we therefore proceeded to test in this report the antitumor effects of BBR in an UF nude mouse xenograft model. When UF nude mice were killed at 7 weeks, tumor weight in controls was 45 ± 7 mg versus 20 ± 3 mg ( P < .05) in the low-dose (5 mg/kg) and 7 ± 3 mg ( P < .01) in the high-dose (10 mg/kg) BBR groups, respectively. Expression of proliferation markers, cell cycle-related genes, and UF-related genes was downregulated in tumors. No unusual behavioral changes and no signs of kidney or liver damage were observed in the animals with BBR treatment. In conclusion, our data suggests that (a) COX2 and PTTG1 are molecular targets for BBR and (b) BBR is potentially an effective and safe anti-UF agent.

Versican Proteolysis by ADAMTS Proteases and Its Influence on Sex Steroid Receptor Expression in Uterine Leiomyoma.

Leiomyomas have abundant extracellular matrix (ECM), with upregulation of versican, a large proteoglycan. We investigated ADAMTS (a disintegrin-like and metalloprotease with thrombospondin type 1 motifs) protease-mediated versican cleavage in myometrium and leiomyoma and the effect of versican knockdown in leiomyoma cells. We used quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting, immunohistochemistry, and RNA in situ hybridization for analysis of myometrium, leiomyoma and immortalized myometrium and leiomyoma cells. Short interfering RNA (siRNA) was used to knockdown versican in leiomyoma cells. This study was performed in an academic laboratory. Study subjects were women with symptomatic or asymptomatic leiomyoma. We quantified messenger RNAs (mRNAs) for versican splice variants. We identified ADAMTS-cleaved versican in myometrium and leiomyoma and ADAMTS messenger RNAs and examined the effect of VCAN siRNA on smooth muscle differentiation and expression of estrogen and progesterone receptors. The women in the symptomatic group (n = 7) had larger leiomyoma (P = 0.01), heavy menstrual bleeding (P < 0.01), and lower hemoglobin levels (P = 0.02) compared with the asymptomatic group (n = 7), but were similar in age and menopausal status. Versican V0 and V1 isoforms were upregulated in the leiomyomas of symptomatic versus asymptomatic women (P = 0.03 and P = 0.04, respectively). Abundant cleaved versican was detected in leiomyoma and myometrium, as well as in myometrial and leiomyoma cell lines. ADAMTS4 (P = 0.03) and ADAMTS15 (P = 0.04) were upregulated in symptomatic leiomyomas. VCAN siRNA did not effect cell proliferation, apoptosis, or smooth muscle markers, but reduced ESR1 and PR-A expression (P = 0.001 and P = 0.002, respectively). Versican in myometrium, leiomyomas and in the corresponding immortalized cells is cleaved by ADAMTS proteases. VCAN siRNA suppresses production of estrogen receptor 1 and progesterone receptor-A. These findings have implications for leiomyoma growth.

MicroRNA-15b regulates reversion-inducing cysteine-rich protein with Kazal motifs (RECK) expression in human uterine leiomyoma.

BackgroundHuman uterine leiomyoma (fibroids; LYO) are the most common benign neoplasms in reproductive-aged women. Dysregulated extracellular matrix and irregular LYO reversion-inducing cysteine-rich protein with Kazal motifs (RECK) expression are thought to be mediated by aberrant microRNA (miR) expression. The relationship of miR-15b and RECK expression in LYO has not been studied.MethodsThe expression levels of miR-15b and RECK were determined by quantitative RT-PCR, Western blot, and immunohistochemistry in cultures derived from commercial primary leiomyoma (cpLYO) and myometrial (cpMYO) cell lines and leiomyoma (pLYO) and myometrium (pMYO) tissue from surgical samples respectively. The relationship between miR-15b and RECK expression in cpLYO and pLYO (compared to their respective myometrial controls) was evaluated following transfection of cell cultures with either miR-15b mimic or inhibitor.ResultsElevated levels of miR-15b were observed in cpLYO (2.82-fold; p = 0.04) and pLYO cell (1.30-fold; p = 0.0001) cultures respectively compared to corresponding MYO cell controls. Following transfection with miR-15b mimic, cpLYO cells (0.62-fold; p < 0.0001) and pLYO cells (0.68-fold; p < 0.0001) demonstrated reduced RECK protein expression. Following transfection with miR-15b inhibitor, cpLYO cells (1.20-fold; p < 0.0001) and pLYO cells (1.31-fold; p = 0.0007) demonstrated elevated RECK protein expression. RECK protein expression was reduced in pLYO tissues (0.73-fold; p < 0.0001) and pLYO (0.47-fold; p = 0.047) cells when compared to the corresponding MYO tissue controls.ConclusionOur findings suggest that miR-15b negatively regulates RECK expression in LYO, and increased miR-15b and decreased RECK expression may contribute to the pathobiology of LYO. The functional significance of miR-15b and RECK expression warrants further investigation as potential therapeutic targets for the treatment of human LYO.

Resveratrol inhibits proliferation of myometrial and leiomyoma cells and decreases extracellular matrix-associated protein expression

Uterine fibroids (leiomyomas) are the most common benign tumours in women during their reproductive age. Hyperplasia of uterine smooth muscle cells and abnormal deposition of extracellular matrix (ECM) are responsible for the development of uterine fibroids. Studies have shown that food rich in phytochemicals can prevent or treat leiomyoma. Therefore, the purpose of this study is to demonstrate the inhibitory effects of resveratrol on uterine fibroid cell growth and ECM-associated protein expression. We found that resveratrol inhibited the proliferation of leiomyoma cell line (ELT3) and uterine smooth muscle cell line (UtSMC) and affected the cell morphology of both cell lines, induced cell cycle arrest at S and G2/M phase, regulated cell apoptosis associated protein expression and induced cell apoptosis, and affected ECM-associated mRNA and protein expression. Our results suggest that resveratrol is potentially effective in preventing the hyperplasia of leiomyoma and uterine smooth muscle cells.

Biological and Mechanistic Characterization of Novel Prodrugs of Green Tea Polyphenol Epigallocatechin Gallate Analogs in Human Leiomyoma Cell Lines.

Uterine fibroids (leiomyomas) are very common benign tumors grown on the smooth muscle layer of the uterus, present in up to 75% of reproductive-age women and causing significant morbidity in a subset of this population. Although the etiology and biology of uterine fibroids are unclear, strong evidence supports that cell proliferation, angiogenesis and fibrosis are involved in their formation and growth. Currently the only cure for uterine fibroids is hysterectomy; the available alternative therapies have limitations. Thus, there is an urgent need for developing a novel strategy for treating this condition. The green tea polyphenol epigallocatechin gallate (EGCG) inhibits the growth of uterine leiomyoma cells in vitro and in vivo, and the use of a green tea extract (containing 45% EGCG) has demonstrated clinical activity without side effects in women with symptomatic uterine fibroids. However, EGCG has a number of shortcomings, including low stability, poor bioavailability, and high metabolic transformations under physiological conditions, presenting challenges for its development as a therapeutic agent. We developed a prodrug of EGCG (Pro-EGCG or 1) which shows increased stability, bioavailability and biological activity in vivo as compared to EGCG. We also synthesized prodrugs of EGCG analogs, compounds 2a and 4a, in order to potentially reduce their susceptibility to methylation/inhibition by catechol-O-methyltransferase. Here, we determined the effect of EGCG, Pro-EGCG, and 2a and 4a on cultured human uterine leiomyoma cells, and found that 2a and 4a have potent antiproliferative, antiangiogenic, and antifibrotic activities. J. Cell. Biochem. 117: 2357-2369, 2016. © 2016 Wiley Periodicals, Inc.

Paricalcitol, a vitamin d receptor activator, inhibits tumor formation in a murine model of uterine fibroids.

We examined the antitumor and therapeutic potentials of paricalcitol, an analog of 1,25-dihydroxyvitamin D3 with lower calcemic activity, against uterine fibroids using in vitro and in vivo evaluations in appropriate uterine fibroid cells and animal models. We found that paricalcitol has potential to reduce the proliferation of the immortalized human uterine fibroid cells. For the in vivo study, we generated subcutaneous tumors by injecting the Eker rat-derived uterine leiomyoma cell line (ELT-3) rat uterine fibroid-derived cell line in athymic nude mice supplemented with estrogen pellets. These mice were administered with vehicle versus paricalcitol (300 ng/kg/d) or 1,25-dihydroxyvitamin D3 (500 ng/kg/d) for 4 consecutive weeks, and the data were analyzed. We found that while both paricalcitol and 1,25-dihydroxyvitamin D3 significantly reduced fibroid tumor size, the shrinkage was slightly higher in the paricalcitol-treated group. Together, our results suggest that paricalcitol may be a potential candidate for effective, safe, and noninvasive medical treatment option for uterine fibroids.

Tranilast, an orally active antiallergic compound, inhibits extracellular matrix production in human uterine leiomyoma and myometrial cells

To determine the effect of tranilast (an antiallergic drug known to suppress fibrosis or to stabilize mast cells) on extracellular matrix production in human leiomyoma and myometrial cells. Laboratory study. University-affiliated laboratory. Seven premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. Cells were treated with tranilast (300μM) for 48hours to measure extracellular matrix and activin-A expression by real-time reverse-transcription polymerase chain reaction and/or immunocytochemistry. The expression of fibronectin, collagen1A1, versican, and activin-A in myometrial and leiomyoma cells. Tranilast decreased fibronectin, collagen 1A1, and versican messenger RNA (mRNA) expression in human primary leiomyoma cell culture. Similar results were found in an immortalized human leiomyoma cell line. Tranilast also decreased the mRNA expression of fibronectin, collagen 1A1, and versican in human primary myometrial cells. The reduced expression of fibronectin and collagen 1 were observed by immunocytochemistry as well. Tranilast also reduced profibrotic growth factor, activin-A mRNA expression in primary myometrial and leiomyoma cells. Our results indicate that tranilast reduced fibronectin, collagen 1A1, versican, and activin-A expression in leiomyoma and myometrial cells, demonstrating its potential as an antifibrotic therapy for human leiomyomas.

Abstract 1305: Activation of G-protein coupled estrogen receptor (GPER) inhibits ELT-3 uterine leiomyoma cell proliferation.

Abstract Uterine leiomyomas are ovarian hormone-responsive tumors originating in the smooth muscle layer of the uterus called the myometrium. These benign tumors require surgery or other clinical intervention in ∼25% of all premenopausal women in the United States. Despite the high frequency and clinical impact of these tumors, the pathogenesis of uterine leiomyomas is not understood. The estrogen-responsive growth of uterine leiomyomas has been well established, however the role of G protein-coupled estrogen receptor (GPER) in leiomyoma cell regulation has not been studied. The membrane bound estrogen receptor GPER, formerly known as GPR30, is a 7-transmembrane G protein-coupled receptor that is activated by 17β-estradiol (E2). A role for GPER has been previously established in cancers that originate in E2-dependent tissues such as the breast and uterus. Other reports show that GPER is expressed in myometrial cells and activation of GPER modulates myometrial contraction suggesting an important regulatory role for this receptor in the myometrium. Based on these reports, we hypothesized that GPER regulates uterine leiomyoma cells and may represent a putative therapeutic target for this tumor type. The Eker rat model-derived uterine leiomyoma (ELT-3) cell line was used as a model system to study GPER in uterine leiomyoma cells. First, the expression of GPER in uterine leiomyoma cells was determined using RNA and protein samples from ELT-3 cells and real-time PCR and immunoblot analysis. Our findings indicate that GPER transcript and protein were expressed in ELT-3 cells. To determine if activation of GPER regulated uterine leiomyoma cell proliferation, ELT-3 cells were treated with G1, a GPER-specific ligand, or vehicle control and counted 5 days after treatment. Data from these experiments indicated that G1 treatment, and potentially GPER activation, reduced the number of cells over time compared to vehicle control. GPER activation is known to result in the accumulation of pERK and intracellular free Ca2+ and such accumulation is used as a biomarker for GPER activation. Upon G1 treatment, pERK and intracellular free Ca2+ were elevated compared to vehicle treatment in ELT-3 cells suggesting that GPER is functional in uterine leiomyoma cells and is activated by G1. In an effort to determine the mechanism of inhibition of uterine leiomyoma cell proliferation by G1 treatment, we hypothesized that treatment of estrogen-stimulated ELT-3 cells with G1 would inhibit the characteristic E2-induced proliferation of this cell type. Growth curve experiments were performed and data obtained in these experiments suggested that G1 treatment resulted in the inhibition of E2-induced ELT-3 cell proliferation. Taken together, these data suggest that GPER is an important regulator of uterine leiomyoma cell proliferation and is a putative target for novel molecular therapeutics for this tumor type. Citation Format: Maryann Castillo, Angelique M. Wimbley, Jacob J. Mayfield, Jenifer C. Lascano, Kevin D. Houston. Activation of G-protein coupled estrogen receptor (GPER) inhibits ELT-3 uterine leiomyoma cell proliferation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1305. doi:10.1158/1538-7445.AM2013-1305

Nanomedicine for Uterine Leiomyoma Therapy

The purpose of this work was to engineer polymeric nanoparticles to encapsulate and deliver 2-methoxyestradiol, a potential antitumor drug for treatment of uterine leiomyoma (fibroids), the most common hormone-dependent pathology affecting women of reproductive age. Encapsulation efficiency and drug release from the nanoparticles were monitored by HPLC. Cell morphology and in vitro cytotoxicity experiments were carried out in a human leiomyoma cell line. The nanoparticles displayed high encapsulation efficiency (>86%), which was verified by differential scanning calorimetry and x-ray diffraction. Excellent long-term stability of the nanoparticles and gradual drug release without burst were also observed. Cellular uptake of fluorescent nanoparticles was confirmed by confocal imaging. The drug-loaded poly(lactic acid) and poly(lactic-co-glycolic acid) nanoparticles induced cytotoxicity in human leiomyoma cells to a significantly greater extent than the free drug at 0.35 µM. This novel approach represents a potential fertility-preserving alternative to hysterectomy.

Down-Regulation of miR-29b Is Essential for the Pathogenesis of Uterine Fibroids.

Uterine leiomyomas (ULs) are the most common neoplasm in women of reproductive age with an accumulative incidence of over 70%. They are believed to arise from the myometrium, but the etiology of this tumor remains unknown. The most notable characteristic of UL is the excess extracellular matrix (ECM), composed mainly of collagen Types I and III. It has been repeatedly reported that many collagen genes (COL1A1, 1A2, 3A1, 5A1, 5A2, and 7A1) are up-regulated in ULs. Since the production of ECM protein is one of the factors that contribute to the tumor growth, it is important to understand the mechanism of collagen over-production in ULs. In addition to collagens, mRNAs of many genes are differentially expressed in the myometrium and ULs. Recent gene expression profiling studies have revealed that many microRNAs (miRNAs) are also aberrantly expressed in ULs. Specifically, miR-29b is consistently found to be down-regulated in ULs in comparison to normal myometrium. Down-regulation of miR-29 has been correlated with an over-expression of collagen in the liver, lung, kidney and cardiac fibrosis. In vitro studies have demonstrated that miR-29b can down-regulate multiple collagen genes including COL1A1, 1A2, 5A1, and 5A2. Moreover, COL3A1 was ranked top among ~1000 potential targets of miR-29b in a target prediction algorism (miSVR) based on the Support Vector Regression model. These observations suggest that the down-regulation of miR-29b may play a role in the overproduction of ECM in ULs. To investigate the role of miR-29b in the pathogenesis of UL, we first established stable human leiomyoma cell lines that restored miR-29b expression levels to normal by lentiviral infection. The miR-29b and control cell lines were then compared in cell proliferation and the expression of potential miR-29b targets including COL1A1 and 3A1. While the growth rate was not affected by an up-regulation of miR-29b, genes for ECM protein including the collagens were significantly down-regulated. It is well known that myometrial and UL cells lose their original characteristics in vitro. Therefore, we further examined the effect of miR-29b expression levels on tumor growth in vivo by utilizing the xenograft model. When the miR-29b level was restored to the level of myometrium, UL cells did not form a solid tumor. Instead, the UL cells grew flat on the surface of the host mouse kidney. Histological analysis indicated that an up-regulation of miR-29b inhibited collagen accumulation. The experiment was repeated with primary human UL cells, and it confirmed the result of the cell line experiment. Thus, the over-production of ECM in UL is due to the down-regulation of miR-29b, which targets mRNAs of multiple collagens including two major collagen genes, COL1A1 and 3A1. Reversely, we generated myometrial cell lines in which miR-29b was permanently knocked down. While the knock-down of miR-29b increased the levels of collagen in vitro, the down-regulation of miR-29b itself was not sufficient to make myometrial cells tumorigenic in vivo. When cell lines were grafted into mice, neither the mir-29b knock-down nor the control cell lines formed tumors. In conclusion, down-regulation of miR-29b is essential for the formation of ULs, but it is not sufficient to transform myometrium into ULs. Administration of miR-29 mimics may prove to be an effective treatment for symptomatic ULs by reducing the tumor volume. This research was supported by NIH/NICHD Grant R01 HD064402.

Leiomyoma growth mediated by disruptions in injury response and angiogenesis

Thrombospondin 1 (TSP1) is an extracellular matrix component that interacts with proteoglycans, integrins, fibrinogen, and fibronectin. It is involved in cell proliferation, apoptosis, angiogenesis inhibition, and activates latent transforming growth factor beta 1. TSP1 is secreted in response to injury, and high expression is restricted to sites of tissue remodeling or injury. Given dependence of leiomyoma formation on both TGF-beta mediated aberrant injury response and angiogenesis, we investigated the role of TSP1 expression in human leiomyoma surgical specimens and in immortalized leiomyoma and patient-matched myometrial cultures. Laboratory study. Surgical specimens and immortalized leiomyoma and patient-matched myometrial cell lines were analyzed using qRT-PCR and western blot analysis. In untreated leiomyoma surgical specimens, there was a 30.2% decrease in TSP-1 mRNA expression when compared to patient matched myometrium. Conversely, TSP-1 protein was nearly undetectable in myometrial tissue, while TSP1 protein expression in leiomyoma surgical specimens remained elevated. We demonstrated a similar pattern in immortalized cell lines. In order to determine if we could alter TSP1 expression in leiomyoma cells to patterns comparable to myometrial cells, we treated leiomyoma cells with the retinoic acid metabolic blocking agent liarozole at concentrations ranging from 10−9M to 10−6M. Compared to untreated leiomyoma cells, leiomyoma cells treated with liarozole demonstrated an additional 35% decrease in TSP1 mRNA expression. Leiomyoma surgical specimens had decreased TSP1 gene expression but much greater TSP1 protein expression compared to patient matched myometrium, a pattern mirrored by in vitro cultures. Treatment with liarozole decreased mRNA expression further. Inhibition of TSP1 expression could disrupt both the fibrotic process and angiogenesis, resulting in potential improvement of leiomyoma bulk symptoms.

Inhibitory effect of curcumin on uterine leiomyoma cell proliferation

Objective. Uterine leiomyomas are the most common gynaecological benign tumour and greatly affect reproductive health and wellbeing. They are the predominant indication for hysterectomy in premenopausal women. Curcumin, a well-known component of turmeric, has been reported to prevent various diseases such as cancer, diabetes and obesity. Previous study reported that curcumin represses the proliferation of several tumour cells. However, there has not been a precise characterisation of the curcumin-induced inhibition of uterine leiomyoma cells. In this study, we investigated the inhibitory effect of curcumin on leiomyoma cells proliferation.Study design. Eker rat-derived uterine leiomyoma cell lines (ELT-3 cells) were used. Cell proliferation was assessed by counting the number of cells and MTS assay. The activation of peroxisome proliferator-activated receptor-gamma (PPARγ) was evaluated by luciferase assay.Results. We found that curcumin significantly inhibited ELT-3 cell proliferation. PPARγ was expressed in ELT-3 cells and curcumin acted as a PPARγ ligand. This inhibitory effect of curcumin was attenuated by the treatment of cells with PPARγ antagonist.Conclusion. These experimental findings in vitro show that the inhibitory effect of curcumin on ELT-3 cell proliferation occurs through the activation of PPARγ. Curcumin may be useful as an alternative therapy for uterine leiomyoma.

Human leiomyoma cell proliferation and extracellular matrix expression is inhibited by integrin signaling

OBJECTIVE: The integrin family of extracellular matrix (ECM) receptors mediates cell-matrix interactions, differentiation and survival. Increased β1 integrin expression has been demonstrated in leiomyomas. Integrins can mediate their activity through RhoA activation. The objective of this study was to determine if functional inhibition of integrin signaling might affect leiomyoma cell proliferation and ECM production.DESIGN: Experimental interventionMATERIALS AND METHODS: Immortalized uterine leiomyoma and myometrial cell lines were incubated with function inhibiting antibodies to α2, α3, α6 and β1 integrins to determine the effect on proliferation. Rho activity was measured using the RhoA G-LISA kit. Proteins were visualized by immunofluorescence. ECM gene expression was evaluated by qRTPCR.RESULTS: After 72hrs of incubation and at 1μg/ml β1 integrin antibody, we observed a 45% growth inhibition of leiomyoma cells compared to 25% inhibition of myometrial cells. Functional inhibition of the α-integrins did not significantly effect proliferation of either cell lines. Leiomyoma cells also showed decreased surface attachment. Higher levels of Rho activity and FAK expression were measured in leiomyoma cells. Following β1 antibody treatment, we observed inhibition of Rho activity (1.5 fold) and serum induced F-actin. Leiomyoma cells expressed a 3-fold higher expression of fibronectin gene compared to myometrial cells. At lower concentration of β1 antibody, leiomyoma cells showed a 3.16+0.32 fold (p<0.05) decreased fibronectin expression. Myometrial cells demonstrated an upregulation of fibronectin gene at all concentrations. Similar expression patterns were observed with collagen 1A1 and versican genes.CONCLUSIONS: Inhibition of β1 integrin signaling resulted in a reduction in leiomyoma cell growth and Rho activity. Furthermore, increased levels of collagen 1A1, versican and fibronectin were dependent upon β1 integrin signaling in leiomyoma cells, suggesting the possibility of a novel pathway to regulate fibroid growth. OBJECTIVE: The integrin family of extracellular matrix (ECM) receptors mediates cell-matrix interactions, differentiation and survival. Increased β1 integrin expression has been demonstrated in leiomyomas. Integrins can mediate their activity through RhoA activation. The objective of this study was to determine if functional inhibition of integrin signaling might affect leiomyoma cell proliferation and ECM production. DESIGN: Experimental intervention MATERIALS AND METHODS: Immortalized uterine leiomyoma and myometrial cell lines were incubated with function inhibiting antibodies to α2, α3, α6 and β1 integrins to determine the effect on proliferation. Rho activity was measured using the RhoA G-LISA kit. Proteins were visualized by immunofluorescence. ECM gene expression was evaluated by qRTPCR. RESULTS: After 72hrs of incubation and at 1μg/ml β1 integrin antibody, we observed a 45% growth inhibition of leiomyoma cells compared to 25% inhibition of myometrial cells. Functional inhibition of the α-integrins did not significantly effect proliferation of either cell lines. Leiomyoma cells also showed decreased surface attachment. Higher levels of Rho activity and FAK expression were measured in leiomyoma cells. Following β1 antibody treatment, we observed inhibition of Rho activity (1.5 fold) and serum induced F-actin. Leiomyoma cells expressed a 3-fold higher expression of fibronectin gene compared to myometrial cells. At lower concentration of β1 antibody, leiomyoma cells showed a 3.16+0.32 fold (p<0.05) decreased fibronectin expression. Myometrial cells demonstrated an upregulation of fibronectin gene at all concentrations. Similar expression patterns were observed with collagen 1A1 and versican genes. CONCLUSIONS: Inhibition of β1 integrin signaling resulted in a reduction in leiomyoma cell growth and Rho activity. Furthermore, increased levels of collagen 1A1, versican and fibronectin were dependent upon β1 integrin signaling in leiomyoma cells, suggesting the possibility of a novel pathway to regulate fibroid growth.

Regulation of PI3K/AKT Pathway by Progestins in Leiomyoma Cells.

Leiomyoma, also known as uterine fibroids, are benign smooth muscle cell tumors that originate from the myometrium. Approximately 70% of pre-menopausal women develop leiomyomas in the United States. Data indicate that progesterone can promote the growth of leiomyomas, however, the pathways involved are relatively unclear. The PI3K/AKT pathway is commonly hyperactivated in human tumors and we have recently shown that leiomyoma cells have increased basal AKT phosphorylation compared to matched myometrial cells. In addition, progestins can rapidly phosphorylate AKT in primary leiomyoma cells. In order to decipher the mechanisms involved in the rapid activation of AKT by progesterone, we utilized both primary and immortalized leiomyoma cells as well as primary myometrial cells and first determined the protein expression of the players in the PI3K/AKT pathway: p85, p110, AKT1, AKT2, and AKT3. The regulatory subunit of PI3K, p85, and catalytic subunit, p110 were expressed in primary and immortalized leiomyoma cells as well as primary myometrial cells. Progestin treatment over 48 hours did not alter p85 or p110 protein levels in these cells. Primary and immortalized leiomyoma cells and primary myometrial cells expressed AKT1 in the presence and absence of serum. AKT2 and AKT3 expression, however, varied depending on the patient in primary leiomyoma cells. AKT2 was expressed in the absence of serum in immortalized leiomyoma cells and primary myometrial cells. Primary myometrial cells also expressed AKT3 in the absence of serum; however, immortalized leiomyoma cells expressed AKT3 only in the presence of serum. Levels of total AKT protein decreased in immortalized leiomyoma cells in response to R5020 or RU486 alone as well as in combination. In primary myometrial cells, total AKT proteins decreased in response to the combination of R5020 and RU486. Finally, we demonstrated that progesterone rapidly phosphorylates AKT, within 15minutes, in the immortalized leiomyoma cell line as it does in the primary leiomyoma cells. Furthermore, the highest level of p-AKT was observed with 10nM and 100nM progesterone. In summary, all three cell types expressed p85, p110 and AKT 1. AKT 2 and AKT 3 expressions were variable, depending on the patient. While long term progestin treatment did not affect p85 or p110 protein levels, total AKT protein was differentially regulated by progestins and serum in the leiomyoma compared to the myometrial cells. In conclusion, not only can progesterone rapidly activate the AKT pathway in leiomyoma cells but it may regulate AKT expression after long term treatment. In addition, AKT isoform expression in primary and immortalized leiomyoma cells may depend on the availability of growth factors and hormones to regulate cell survival. Research supported by a grant from the Friends of Prentice. (poster)