Normal Uterine Smooth Muscle Cells Research Articles

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.

Abstract 4130: Improvement of drug efficacy of polo-like kinase 4 inhibitor in the treatment of uterine leiomyosarcoma

Abstract Introduction: Uterine leiomyosarcoma (Ut-LMS) is aggressive. Recurrence usually develops in 50 - 70% of patients and effective chemotherapy regime are limited. Polo-like kinase 4 (PLK4) is vital modulator of centriole duplication and cell mitotic progression. Inhibition of PLK4 disrupts mitosis, which leads to polyploidy, further genomic instability, mitotic catastrophe, and cell death. Recently, CFI-400945, a PLK4 inhibitor, has been found effective in preclinical studies in the treatment of several malignancies but its effectiveness on Ut-LMS has not been investigated. In phase I studies, CFI-400945 was well tolerated although toxicity, primarily reversible neutropenia, was observed at higher doses. Ataxia-telangiectasia mutated (ATM) is a key kinase in the DNA damage response pathway. AZD0156, an ATM kinase inhibitor, has recently been proposed to be used with other DNA damaging agents in cancer treatments. We explored the effectiveness of a combined approach with CFI-400945 and AZD0156 in Ut-LMS. Methods: Ut-LMS cell lines SK-UT-1, SKN and SK-LMS-1 cultured in-vitro were treated with CFI-400945 and AZD0156. PLK4 gene expression in these cell lines was also determined by RT-qPCR assays. Cell proliferation was examined using the sulforhodamine B (SRB) assay while apoptosis was examined by Annexin V-PI and caspase 3/7 assays. Immunofluorescence confocal centrosome quantification and flow cytometry PI cell cycle analysis were used to detect centrosome overduplication and generation of aneuploidy cells. The effect of gene expression in normal uterine smooth muscle cells and Ut-LMS cell lines treated with CFI-400945 was performed by RNA-Seq. DNA damage was detected using neutral comet assay. Results: Ut-LMS cell lines expressed high levels of PLK4 and were highly sensitive (IC50 in nM range) to CFI-400945. Centrosome overduplication, aneuploidy cells, and cell growth inhibition were observed in a dose-dependent manner, and prominent apoptosis was observed at 48 hours after drug treatment. Gene set enrichment analysis on RNA-Seq data indicated that cells were under genotoxic stress after CFI-400945 treatment while comet assay verified DNA damage. Cell arrest and apoptosis with CFI-400945 was further enhanced by the addition of AZD0156. Combined treatment of CFI-400945 and AZD0156 produced synergistic antineoplastic effects in all three Ut-LMS cell lines, with combination indices <1. DNA damage was also enhanced in the combined treatment group versus the single treatment group. Conclusion: The PLK4 inhibitor CFI-400945 was a potent treatment for Ut-LMS in vitro. This effect was further augmented when combined with the DNA damage response pathway inhibitor AZD0156. The synergistic effect of these agents is promising, and should be investigated further in the treatment of Ut-LMS Citation Format: Hok Yeung Horace Lee, Kin Long Chow, Ho Shing Wong, Tsz Yan Chong, Ka Yu Tse, Mark R. Bray, Tak Wah Mak, Philip PC Ip. Improvement of drug efficacy of polo-like kinase 4 inhibitor in the treatment of uterine leiomyosarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4130.

NF-κB inhibits the differentiation of hysteromyoma cells by reducing myocardin expression.

To investigate the effect of nuclear factor-kappaB (NF-κB) on the myocardin-mediated differentiation of hysteromyoma cells. Expression levels of myocardin in hysteromyoma cells from patients with hysteromyoma were detected. Normal uterine smooth muscle cells were used as control group. Overexpression of myocardin in hysteromyoma cells was achieved through lentivirus infection. Changes in expression levels of uterine smooth muscle cell maker p21, p57, Cyclin D1, PCNA, SM22α, and αSMA were detected. Hysteromyoma cells with lentivirus infection were stimulated by lipopolysaccharide (LPS), and changes in expression levels of myocardin were detected. Compared with normal uterine smooth muscle cells, the expression level of myocardin in hysteromyoma cells was extremely low, or even undetectable, and expression levels of smooth muscle cell differentiation markers were also minimal, and cells were in the de-differentiated state. Expression of exogenous myocardin can improve the expression of smooth muscle cell differentiation markers to induce cell re-differentiation. LPS stimulation can activate NF-κB to inhibit myocardin expression, thereby inducing cell dedifferentiation. NF-κB can inhibit the differentiation of hysteromyoma cells by decreasing the expression level of myocardin.

A High Concentration of Genistein Induces Cell Death in Human Uterine Leiomyoma Cells by Autophagy.

Genistein, an estrogenic, soy-derived isoflavone, may play a protective role against hormone-related cancers. We have reported that a high concentration of genistein inhibits cell proliferation and induces apoptosis in human uterine smooth muscle cells, but not in leiomyoma (fibroid) cells. To better understand the differential cell death responses of normal and tumor cells to a high concentration of genistein, we treated uterine smooth muscle cells and uterine leiomyoma cells with 50 μg/ml of genistein for 72 h and 168 h, and assessed for mediators of apoptosis, cytotoxicity and autophagy. We found that leiomyoma cells had increased protection from apoptosis by expressing an increased ratio of Bcl-2: bak at 72 h and 168 h; however, in smooth muscle cells, the Bcl-2: bak ratio was decreased at 72 h, but significantly rebounded by 168 h. The apoptosis extrinsic factors, Fas ligand and Fas receptor, were highly expressed in uterine smooth muscle cells following genistein treatment at both time points as evidenced by confocal microscopy. This was not seen in the uterine leiomyoma cells; however, cytotoxicity as indicated by elevated lactate dehydrogenase levels was significantly enhanced at 168 h. Increased immunoexpression of an autophagy/autophagosome marker was also observed in the leiomyoma cells, although minimally present in smooth muscle cells at 72 h. Ultrastructurally, there was evidence of autophagic vacuoles in the leiomyoma cells; whereas, the normal smooth muscle cells showed nuclear fragmentation indicative of apoptosis. In summary, our data show differential cell death pathways induced by genistein in tumor and normal uterine smooth muscle cells, and suggest novel cell death pathways that can be targeted for preventive and intervention strategies for inhibiting fibroid tumor cell growth in vivo.

Berberine inhibits the proliferation of human uterine leiomyoma cells

To determine whether berberine (BBR), a naturally occurring plant-derived alkaloid, inhibits the proliferation of human uterine leiomyoma (UtLM) cells. Laboratory research. Laboratory. UtLM and normal human uterine smooth muscle (UtSMC) cell lines. Treatment with [1] BBR (10, 20, and 50 μM), [2] BBR (20 and 50 μM) and/or 17β-estradiol (E2; 10 and 100 nM), and [3] BBR (20 and 50 μM) and/or progesterone (P4; 10 and 100 nM) for 24 or 72 hours. Cell proliferation, cell cycle, apoptosis, and related genes expression were determined. BBR inhibited UtLM cell proliferation by inducing G2/M cell cycle arrest and apoptosis. Cell cycle G2/M phase-related genes were altered by BBR treatment: the expression of cyclin A1, cyclin B1, and Cdk1 were down-regulated, while Cdk4, p21, and p53 were up-regulated. BBR-treated cells stained positively for annexin V and manifested increased BAX expression. E2- and P4-induced UtLM cell proliferation was blocked by BBR treatment. In marked contrast, even the highest concentration of BBR (50 μM) did not influence cell proliferation in UtSMC cells. BBR selectively inhibits cellular proliferation and blocks E2- and P4-induced cell proliferation in UtLM but not in normal UtSMC cells. In addition, BBR did not demonstrate cytotoxicity effects in normal human UtSMCs. Our results suggest BBR could be a potential therapeutic agent for the treatment of uterine leiomyoma.

An endocrine-disrupting chemical, fenvalerate, induces cell cycle progression and collagen type I expression in human uterine leiomyoma and myometrial cells

Fenvalerate (Fen), widely used for its high insecticidal potency and low mammalian toxicity, is classified as an endocrine-disrupting chemical. Recently, Fen has received great attention for its adverse effects on human reproductive health. In this study, we found that Fen (10 μM) had a stimulatory effect on the growth of both cell lines at 24 h compared with controls by MTS ( p < 0.01) and BrdU ( p < 0.01) assays in hormonally responsive uterine leiomyoma (UtLM) cells and normal uterine smooth muscle cells (UtSMC). Flow cytometry results showed that Fen enhanced the escape of cells from the G 0–G 1 checkpoint and promoted progression of both cell types into the S phase. An Annexin V assay showed that Fen had an anti-apoptotic effect on both cell types. By Real-time PCR, we found that collagen I mRNA expression increased ( p < 0.05) in Fen-treated cells compared to controls, although it was greater in UtLM tumor cells. Accordingly, Fen increased ( p < 0.05) collagen I protein levels in both cell lysate and supernatant when compared to controls. To further test the mechanism of Fen's effects, transactivation and competitive binding assays were done. The results showed Fen did not significantly stimulate luciferase activity at concentrations of 0.1 μM, 1.0 μM or 10.0 μM in either of the cell types. Competitive binding assays revealed that the affinity of Fen binding to estrogen receptors (ERs) was non-detectable compared to E 2. Our data show that Fen can stimulate the growth of both UtLM cells and UtSMC, which involves a combination of enhanced cell cycle progression and inhibition of apoptosis. Also this compound can increase collagen I expression, at both mRNA and protein levels. Interestingly, the ER is less likely involved in either the hyperplasia or extracellular matrix (ECM) overproduction induced by Fen. Our results indicate that Fen exposure could be considered a novel risk factor for uterine fibroids through molecular mechanisms that do not directly involve the ERs.

Estrogen-induced changes in IGF-I, Myb family and MAP kinase pathway genes in human uterine leiomyoma and normal uterine smooth muscle cell lines

Many studies have implicated numerous hormones, growth factors, cytokines and other signal transduction molecules in the pathogenesis of uterine leiomyoma. Estrogen and estrogen-related genes are thought to play a key role in the growth of uterine leiomyomas, but the molecular mechanisms are unclear. In an attempt to investigate various pathways that might be involved in estrogen-regulated uterine leiomyoma growth as well as to identify any novel effector genes, microarray studies comparing estrogen-treated uterine leiomyoma cells (UtLM) and normal myometrial cells to untreated cells were performed. Several genes were differentially expressed in estrogen treated UtLM cells, including insulin-like growth factor-I (IGF-I) and others potentially involved in the IGF-I signalling pathway, specifically genes for A-myb, a transcription factor which promotes cell cycle progression and for MKP-1, a dual specificity phosphatase that dephosphorylates mitogen-activated protein kinase. IGF-I and A-myb were up-regulated in estrogen-treated cells while MKP-1 was down-regulated. Two other cell cycle promoting genes, c-fos and myc, were also down-regulated in estrogen treated UtLM cells. These genes are typically up-regulated in response to estrogen in some cells, notably breast epithelial cells, yet consistently have lower expression levels in uterine leiomyoma tissue when compared to autologous myometrium. Our results demonstrate some novel genes that may play a role in the growth of uterine leiomyoma, strengthen the case for involvement of the IGF-I pathway in the response of UtLM to estrogen and corroborate evidence that uterine smooth muscle cells respond to estrogen with a different gene expression pattern than that seen in epithelial cells.

Smooth muscle differentiation in human myometrium and uterine leiomyoma.

Smooth muscle differentiation has been analysed in human myometrium and leiomyoma by Western blotting with antibodies to smooth muscle specific proteins. No differences in the expression of h-caldesmon, metavinculin, desmin, alpha-smooth muscle actin and calponin were observed. The technique of two-dimensional gel electrophoresis was used, therefore, to further analyse differences between normal smooth muscle cells and their neoplastic counterparts. By comparing the protein patterns of normal myometrium and leiomyoma, it was possible to identify a protein with a molecular weight of approximately 27 kD that is selectively expressed in normal uterine smooth muscle cells. This protein proved to be a low molecular weight variant of calponin, a smooth muscle specific protein of as yet unknown function. Its immediate downregulation in tissue culture of normal myometrium points to a possible role in the process of dedifferentiation.