tretinoin and Leiomyoma

Leiomyomas, monoclonal tumors developed by the transformation of myometrium somatic stem cells, are a major health concern that can severely impair quality of life. Pathological alterations of signaling pathways have been recognized as a key feature in a variety of human diseases. Our objective was to analyze treatment with all-trans-retinoic acid (ATRA) by suppression of the phosphoinositide 3-kinase (PI3K) pathway on growth, signaling pattern and interactions among PI3K/B-cell lymphoma 2 (Bcl2)/retinol leiomyoma proteins.. Cultures of paired myometrium and leiomyoma cells from premenopausal women undergoing hysterectomy were collected. Western blot and analysis of variance were used for analysis.. Significant differences were detected between treatment with ATRA alone or with LY294002 (a PI3K growth suppressor) in response to treatment and among cell samples and cell numbers. Leiomyoma cells were less affected. Immunochemical analysis of signaling patterns demonstrated that treatments affected most of the examined protein levels differently. Significant differences between the cell type responses to treatment in pyruvate phosphate dikinase 1 (pPDK1), Bad and pβ-catenin levels were identified. The pβ-catenin level showed highly significant interaction between response to treatment and cell type.. ATRA treatment on PI3K pathway suppression significantly affected growth, signaling pattern and interactions among PI3K/Bcl2/retinol proteins involved in the growth, survival and apoptosis of leiomyomas. Interpretation of our results suggests that increasing knowledge of the role of signaling interplay in the pathogenesis of leiomyomas may present an opportunity to use specific signal transduction inhibitors for treating and preventing this disorder.

Topics: Adult; bcl-Associated Death Protein; beta Catenin; Cell Line, Tumor; Chromones; Female; Humans; Hysterectomy; Leiomyoma; Middle Aged; Morpholines; Myometrium; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Premenopause; Protein Serine-Threonine Kinases; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Signal Transduction; Tretinoin; Uterine Neoplasms

Uterine fibroids are a prevalent gynaecological condition in reproductive-aged women and are the commonest reason for hysterectomy. The cellular composition of clonal fibroids are heterogeneous, with phenotypically dissimilar cells that include smooth muscle cells (SMC), vascular SMC (VSMC) and fibroblasts. The aim of our study was to investigate genes that are commonly differentially expressed between fibroid and myometrial whole tissues in phenotypically different sub-populations of cells isolated from fibroid and myometrium. Genes to be investigated by fluorescence-activated cell sorting, quantitative real-time PCR and immunocytochemistry include transforming growth factor β (TGFB) and retinoic acid (RA) signalling families and steroid hormone receptors. We hypothesised that each cell population isolated from fibroid and myometrium would differ in the expression of fibroid-associated genes. We demonstrated that phenotypically different cellular constituents of uterine fibroids differentially express cellular RA-binding protein 2 (CRABP2), progesterone receptor B (PRB) and TGFB receptor 2 mRNA in fibroid-derived cells of VSMC and SMC phenotype. CRABP2 mRNA was also differentially expressed in fibroblasts and VSMC sub-populations from within clonal fibroid tumours. We conclude that differential regulation of RA, TGFB and PR pathway transcription occurs in fibroid-associated SMC and -fibroblasts and that investigation of paracrine interactions between different cell types within the fibroid microenvironment provides an important new paradigm for understanding the pathophysiology of this common disease.

Topics: Female; Fibroblasts; Gene Expression Regulation; Gonadal Steroid Hormones; Humans; Leiomyoma; Middle Aged; Muscle, Smooth; Muscle, Smooth, Vascular; Myometrium; Paracrine Communication; Phenotype; Receptors, Progesterone; Receptors, Retinoic Acid; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factors; Tretinoin

To study the influence of liarozole on leiomyoma cell proliferation and extracellular matrix (ECM) gene expression in immortalized leiomyoma cells.. Laboratory study.. University hospital.. None.. Tissue culture, real-time reverse transcription-polymerase chain reaction, Western blot.. Proliferation, messenger RNA (mRNA), and ECM protein expression.. Proliferation of leiomyoma cells was inhibited by treatment with liarozole at suprapharmacologic concentrations. The mRNA and protein expression of COL1A1, COL4A2, versican, fibromodulin, and fibronectin was increased in untreated leiomyoma cells compared with untreated patient-matched myometrial cells. Extracellular matrix mRNA expression was decreased in a dose-dependent manner in leiomyoma cells treated with pharmacologic concentrations of liarozole. In addition, myometrial cells treated with liarozole demonstrated no statistically significant alteration in ECM regulation.. Liarozole inhibited ECM protein production at pharmacologic concentrations in immortalized human leiomyoma cells. Retinoic acid metabolic blocking agents represent a potential therapeutic drug family for human leiomyomas.

Topics: Antineoplastic Agents, Hormonal; Cell Proliferation; Dose-Response Relationship, Drug; Extracellular Matrix; Extracellular Matrix Proteins; Female; Humans; Imidazoles; Leiomyoma; Tretinoin; Tumor Cells, Cultured; Uterine Neoplasms

To detect changes induced by all-trans-retinoic acid (ATRA) on the expression and activation of target proteins of the retinoic acid (RA) and PI3K/Akt pathways involved in leiomyoma growth.. A study on human tissue cultures.. Hadassah University Hospital.. Premenopausal women with uterine leiomyomas.. Paired cultures of normal myometrium and leiomyomas, from women undergoing hysterectomy, were obtained.. The effect of ATRA was examined on the expression and phosphorylation of relevant RA, PI3K/Akt, and Bcl2 proteins (immunochemical analysis), cell proliferation, cell cycle distribution, and apoptosis.. Applying our cell culture model, we demonstrated that ATRA induced changes in the expression and activation of the RA and PI3K/Akt pathway proteins in leiomyoma cells, with significant increases of alcohol dehydrogenase 1 and cyclin D2 protein levels. In part of the leiomyoma cells, ATRA induced a relative increase of Bax (proapoptotic) as well as a relative decrease of phosphorylated glycogen synthase kinase 3β (proapoptotic).. Our results highlight the involvement of ATRA in the RA and PI3K/Akt pathways, whose specific signaling products may influence the outcome of leiomyoma growth by regulating cell proliferation, apoptosis, and survival. These results might be useful for the on-going research into alternative methods for treating and preventing this disorder.

Topics: Adult; Apoptosis; Cell Cycle; Cell Proliferation; Cells, Cultured; Drug Evaluation, Preclinical; Female; Humans; Leiomyoma; Matched-Pair Analysis; Middle Aged; Models, Biological; Phosphatidylinositol 3-Kinases; Receptors, Retinoic Acid; Signal Transduction; Tretinoin; Uterine Neoplasms

Uterine leiomyomas are clinically significant tumours that may develop due to an altered differentiation pathway. We have previously identified a dysregulated retinoic acid (RA) pathway that reduced retinoic exposure in human leiomyoma surgical specimens, and have shown that the leiomyoma phenotype was characterized by excessive and disorganized extracellular matrix (ECM).. The goal of this study was to determine the impact of RA exposure on the disrupted ECM phenotype of leiomyomas.. Study of immortalized and molecularly confirmed cells generated from surgical specimens of spontaneous uterine leiomyoma and matched myometrium.. Immortalized leiomyoma and myometrial cells retained the molecular characteristics of their progenitor tissue. Proliferation of leiomyoma cells was inhibited by all-trans retinoic acid (ATRA). Furthermore, there was a dose-dependent decrease in soluble extracellular collagen protein in ATRA-treated leiomyoma cells. Exposure of leiomyoma cells to ATRA resulted in a dose-dependent inhibition of templates for specific ECM protein production including collagen 1, collagen 4, fibronectin and versican. Notably, expression levels in treated leiomyoma cells approached those found in myometrial cells. These mRNA alterations translated into altered protein. Down-regulation was also observed among the RA pathway genes such as CYP26A1 with exposure to ATRA. Finally, ATRA down-regulated TGF-beta3 mRNA expression and the TGF-beta regulated genes in leiomyoma cells.. Exposure of leiomyomas to ATRA down-regulated cell proliferation, ECM formation, RA metabolism and TGF-beta regulation, suggesting that RA exposure can alter the leiomyoma phenotype to one that more closely approximates normal myometrium.

Topics: Cell Differentiation; Cell Proliferation; Cells, Cultured; Collagen; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Extracellular Matrix; Female; Gene Expression Regulation, Neoplastic; Humans; Leiomyoma; Myometrium; Phenotype; Signal Transduction; Tretinoin; Uterine Neoplasms

Despite the fact that uterine fibroids are the most common benign tumors in women, their etiology is poorly understood. We have previously shown that multiple members of the retinoic acid (RA) pathway have altered expression in fibroids compared with normal myometrium. The aims of the present study were: to investigate regulation of genes involved in the RA pathway in vitro; and to identify genes that can be used as markers to distinguish myometrial and fibroid smooth muscle cells in culture.. We demonstrate here for the first time that differential expression of aldehyde dehydrogenase 1 (ALDH1) between fibroids and myometrium is maintained in cell culture (without endothelial cells), and that this gene is differentially regulated by retinoids in myometrial compared with fibroid cells. RA and retinol also regulate expression of ADH1, cellular retinol binding protein 1 and cellular RA binding protein 2 in fibroid and myometrial cells. We show that many of the RA pathway genes tested maintain expression levels and differences in vitro. We also identify nine genes that are differentially expressed between myometrium and fibroids and maintain these differences and expression levels in cultured cells isolated from the same tissues. These genes can be used as markers to distinguish myometrial and fibroid cells in culture.. Based on these findings, we propose that the RA pathway has an important and possible causative role in fibroid growth, as evidenced by the large number of genes with significantly altered expression in uterine fibroids that can be regulated by RA.

Topics: Adult; Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Chemokines; Down-Regulation; Female; Humans; Intercellular Signaling Peptides and Proteins; Isoenzymes; Leiomyoma; Middle Aged; Muscle, Smooth; Myometrium; Receptors, Retinoic Acid; Retinal Dehydrogenase; Retinoids; Retinol-Binding Proteins, Cellular; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Up-Regulation; Vitamin A

An alteration of the retinoid pathway can influence the development of uterine leiomyomas in animal models, and retinoids have shown efficacy in inhibiting the growth of this benign tumor both in vitro and in vivo. However, the underlying mechanisms and biological implications are unclear. The present study was based on the demonstration of an accumulation of full-length retinoid X receptor alpha (RXRalpha) in leiomyomas that was not associated with a modification of its gene expression. This accumulation was shown to increase the transcription of the RXR-responsive gene cellular retinoic acid binding protein II (CRABP-II) and to be linked to the cellular redistribution of the receptor and to its retarded degradation via the ubiquitin/proteasome pathway. Accordingly, treatment with a specific proteasome inhibitor but not with protease inhibitors strongly inhibited the degradation of full-length RXRalpha in cells deriving from both myometrium and leiomyoma, but the formation of RXRalpha/ubiquitin conjugates was differentially regulated between the two cell types. Moreover, full-length RXRalpha accumulated in leiomyomas was abnormally phosphorylated at serine/threonine residues relative to myometrial tissue. The ligand to RXRalpha, 9-cis-retinoic acid, induced the receptor breakdown in smooth muscle cells deriving from both normal and tumor tissue, whereas a MAPK-specific inhibitor was able to reduce RXRalpha levels only in leiomyoma cells. These results suggest that switching of the ubiquitin/proteasome-dependent degradation of RXRalpha by phosphorylation in leiomyomas may be responsible for the accumulation of the receptor and the consequent dysregulation of retinoic acid target genes. The ability of retinoids to modify this molecular alteration may be the rationale for their use in the treatment of leiomyomas.

Topics: Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; Leiomyoma; Myocytes, Smooth Muscle; Phosphorylation; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Protein Transport; Retinoid X Receptor alpha; Transcriptional Activation; Tretinoin; Uterine Neoplasms

To analyze expression of the retinoic acid signaling pathway genes that are involved in retinol metabolism, transport, transcriptional activation, and transcriptional products in spontaneous human leiomyomas.. Laboratory study of human leiomyoma and patient-matched myometrial tissue.. Eight women undergoing hysterectomy for symptomatic leiomyomas.. Confirmation of an altered retinoic acid pathway analyzed by microarray, real time reverse transcription-polymerase chain reaction, Western blot, immunohistochemistry, and high-performance liquid chromatography (HPLC).. Gene and protein expression.. Regardless of patient demographics and leiomyoma location and size, we found decreased expression of the major genes involved in retinoic acid pathway including alcohol dehydrogenase-1 (-3.97- +/- 0.03-fold), aldehyde dehydrogenase-1 (-3.1- +/- 0.07-fold), cellular retinol binding protein-1 (-2.62- +/- 0.04-fold), and cellular retinoic acid binding protein-1 (-2.42- +/- 0.20-fold). Cytochrome P450 (CYP 26A1), which is responsible for retinoic acid metabolism, was highly up-regulated in leiomyomas (+5.4- +/- 0.53-fold). Nuclear receptors demonstrated a complex pattern of under-expression (RARalpha, RARbeta, RXRalpha, RXRgamma) and over-expression (RARgamma, RXRbeta) at both the mRNA and protein level. Differences in protein amounts were confirmed by Western blot. Finally, a reduced amount of cellular ATRA and 9-cis retinoic acid was confirmed by HPLC in leiomyomas compared with myometrial tissues.. Molecular alterations in the retinoic acid pathway of leiomyomata result in a decrease in retinoic acid exposure.

Topics: Adult; Female; Gene Expression Regulation, Neoplastic; Humans; Hysterectomy; Immunohistochemistry; Leiomyoma; Menstrual Cycle; Middle Aged; Myometrium; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Tretinoin; Uterine Neoplasms

Fibroids are benign neoplasms of myometrial smooth muscle cells (SMC). Despite being the most common tumor in humans, their etiology is poorly understood. Recent microarray studies have demonstrated that multiple members of the retinoid pathway are differentially expressed between myometrium and fibroids. The aim of this present study was to investigate gene expression of members of the retinoid pathway in matched myometrium and fibroids. We have demonstrated differential gene expression of two binding proteins [cellular retinol-binding proteins (CRBP) 1 and 2], three enzymes [alcohol dehydrogenase 1 (ADH1), aldehyde dehydrogenase (ALDH1) and retinol dehydrogenase (RODH)] and two receptors [retinoid X receptors (RXR) alpha and gamma] involved in the retinoid pathway by real-time PCR. There were no differences in gene expression for retinoid receptors RARalpha, beta, gamma and RXRbeta, and for the metabolizing enzyme cytochrome P450, family 26 subfamily A. We confirmed results for ADH1, ALDH1, CRBP1 and CRABP2 at the protein level by western blot. Using immunohistochemistry these proteins were mostly localized to myometrial and fibroid SMC. An exception to this was ALDH1 protein, which displayed strong staining localized to cells of the connective tissue, presumably fibroblasts, with a striking differential expression pattern between myometrium and fibroids. These results demonstrate that the retinoid pathway is altered in fibroids when compared with normal myometrium and specifically identify ALDH1 in fibroid fibroblasts. These alterations can lead to aberrant retinoic acid (RA) production and signaling, and alter the expression of RA target genes, which may be an important step in fibroid development.

Topics: Adult; Alcohol Dehydrogenase; Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Female; Gene Expression; Histone-Lysine N-Methyltransferase; Humans; Isoenzymes; Leiomyoma; Middle Aged; Myometrium; Retinal Dehydrogenase; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Tretinoin; Uterine Neoplasms

Primary cultures of human uterine smooth muscle cells have been widely used as a model system to evaluate agents that may play a role in the regulation of both normal and abnormal proliferative responses. We have used this in vitro system to determine if human uterine smooth muscle cells are responsive to treatment with a potent natural derivative of vitamin A, all-trans retinoic acid (ATRA). These studies were also designed to determine if there is a difference in retinoid responsiveness between normal smooth muscle and adjacent leiomyoma (a benign tumor of uterine smooth muscle). When cells were cultured in the presence of ATRA, a dose-dependent inhibition in proliferation was observed. This inhibition in proliferation was accompanied by an alteration in smooth muscle cell morphology. Both the inhibition in proliferation and the altered morphology were reversible when ATRA treatment was discontinued. Responsiveness to retinoids is determined, in part, by the expression of ligand-specific receptors belonging to the steroid/thyroid superfamily (RARs and RXRs); we have therefore identified the pattern of retinoid receptor transcript expression in human uterine smooth muscle cells. The data indicate that human uterine smooth muscle cells express retinoic acid receptors RAR alpha, beta, and gamma, and retinoid X receptors RXR alpha and beta. No difference in retinoid responsiveness or in the pattern of retinoid receptor expression was observed between normal smooth muscle and adjacent leiomyoma. This is the first observation of an antiproliferative effect of ATRA in uterine smooth muscle cells and the first report of retinoid receptor expression patterns in this cell type. Since retinoids are common pharmacologic tools in the treatment of a wide variety of hyperproliferative disorders, these observations may have both therapeutic and toxicologic implications.

Topics: Cell Division; Cell Size; Cells, Cultured; Female; Gene Expression Regulation; Humans; Leiomyoma; Microscopy, Phase-Contrast; Muscle, Smooth; Nucleic Acid Hybridization; Receptors, Retinoic Acid; Retinoid X Receptors; Ribonucleases; RNA, Antisense; RNA, Messenger; Transcription Factors; Tretinoin; Tumor Cells, Cultured; Uterus