Study Of The Effects Of Neonatal Exposure To Genistein On The Development Of Uterine Leiomyomas

Uterine leiomyomas (UtLMs), also called fibroids, are the mostcommon reproductive tract neoplasm arising from the smooth musclewall of uterus and the leading cause of hysterectomy in premenopausalwomen. With the recognition of the ubiquitous presence of many of theenvironmental estrogens, the high incidence of uterine leiomyoma (UtLM)in women has called into question the potential influence ofenvironmental estrogens in the pathogenesis of these tumors. In humansand experimental animals, it has been well established that exposure tothe synthetic estrogen diethylstilbestrol (DES) at low doses during criticalperiods of reproductive tract differentiation permanently alters estrogentarget tissues and results in long-term abnormalities such as UtLMs whichare not manifested until later in life. Developmental origin of UtLMs hasalso been suggested by exposure to other environmental estrogens at equal estrogenic doses similar to low doses of DES. Among theseenvironmental estrogens, genistein (Gen), a naturally occurringphytoestrogen, is found in high levels in soy products known to bepresent in human diet and is likely substantial component of vegetariandiets. The effect of Gen on pregnant women and infancy is of particularconcern in recent years. In CD-1 mice, neonatal Gen exposure atenvironmentally relevant doses (0.5-50 mg per kg per d) causes adverseconsequences on the developing female reproductive system includingabnormal ovarian development, altered reproductive function and uterineneoplasia later in life. However, the long-term consequence regarding therisk of developing UtLMs is poorly investigated. Therefore, the specificaims of our study are to: (1) Elucidate the molecular mechanism(s) thatmight be involved in the development of UtLM after exposure to Genneonatally in CD-1 mice. (2) Test the effects of Gen on human UtLMcells and normal uterine smooth muscle cells (UtSMC) in vitro. (3)Identify candidate genes that are differentially expressed in UtLM cellsafter treatment with low and high concentrations of Gen by cDNAmicroarray analysis, thus to elucidate the molecular mechanisms bywhich exposure to Gen may impact the development of these tumors. (4)Elucidate the mechanisms that involved in the hyperresponsiveness ofUtLM cells compared to UtSMC by treated with a low concentration ofGen. These experiments will yield novel insights into the molecularmechanisms responsible for the development of uterine leiomyomas byendogenous and exogenous hormones, and increase our understanding ofthe potential mechanisms by which exposure to endocrine disruptorscould contribute to the development of this disease.SectionⅠ: Neonatal genistein exposure increases estrogen receptoralpha and insulin-like growth factor-Ⅰreceptor expression in thedevelopment of uterine leiomyoma Outbred CD-1 mice were treated neonatally on Days 1-5 with thephytoestrogen, genistein (Gen) 0.5, 5 or 50 mg per kg per day (refereed toas Gen-0.5, Gen-5 and Gen-50), and uteruses were collected at months 12and 18. Hematoxylin and eosin (H&E) staining showed tumors andtypical histomorphologic characteristics of UtLM in Gen-5 and Gen-50treated groups at 12 and 18 months, respectively, but not in Gen-0.5treated and control groups. The results of the immunohistochemicalstaining demonstrated that both of the protein expression of estrogenreceptor-alpha (ERα) and phosphorylation of ERαwas higher in uterineleiomyoma cells than that of matched myometrium. Furthermore, theimmunoexpression of insulin-like growth factor-Ⅰ(IGF-Ⅰ),IGF-Ⅰreceptorand phosphorylated mitogen-activated protein kinase (MAPK) was alsofound increased in tissues, which were identified as UtLMs, compared tonormal myometrium tissues. Conclusions: Neonatal exposure of Geninduced UtLMs later in CD-1 mice and this effect was dose-dependent.Development of uterine leiomyoma was associated with the increasedprotein expression of ERα,phosphorylation of ERα,IGF-Ⅰ,IGF-Ⅰreceptor and activation of MAPK in uterine leiomyoma cells, suggestingthat both ERαand IGF-Ⅰreceptor signaling pathways might be disruptedby early Gen exposure, and both of them play an important role in thegrowth of UtLM. Therefore, given that human infants are exposed to highlevels of Gen in soy-based foods, this study indicates that the use ofsoy-based infant formulas in the absence of medical necessity and themarketing of soy products designed to appeal to children should beclosely examined. SectionⅡ: In vitro study: the effects of genistein on human uterineleiomyoma cells and normal uterine smooth muscle cellsDue to the dietary exposure of Gen to women and the estrogenresponsiveness of UtLMs "fibroids", we evaluated the effects of Gen onhuman UtLM cells versus normal UtSMC in vitro. We treated humanUtLM cells and UtSMC with 0.001-50μg/ml of Gen. Light microscopywas used to determine the effects on morphology. Cell proliferation wasassessed using a MTS-based assay and proliferating cell nuclear antigen(PCNA) immunocytochemistry. Flow cytometry was used to quantitatecells in S-phase or those undergoing apoptosis. A fluorometric assay andconfocal microscopy were used to detect caspase-3 activity and apoptoticbodies, respectively. The main results are as follows:1. Low doses (0.001-1μg/ml) of Gen had a significant (p＜0.01)stimulatory effect on the growth of UtLM cells, maintained normalelongated appearance of the cells, increased PCNA labeling andpercentage of UtLM cells in S-phase, but not UtSMC.2. Higher doses (10-50μg/ml) of Gen significantly (p＜0.01) inhibitedthe growth of UtSMC and UtLM cell, adversely affected themorphology of UtLM cells and UtSMC, decreased PCNA labeling,increased the percentage of cells in the G2/M phase with decreasednumber of cells in the G1 phase compared to low dose and controlgroups. Further, increased caspase-3 activity and induced apoptosiswere found in both cell types.Conclusions: The effect of Gen is dose-dependent in UtLM cells andUtSMC. Lower doses of Gen elicit proliferative effects on UtLM cellsonly; whereas, higher doses alter the morphology, inhibit proliferation,increase caspase activity and apoptosis in both cell types, with the lattertwo effects being more extensive in UtSMC. SectionⅢ: Insights from toxicogenomics: High-dose of genisteindown-regulates TGF-beta pathway genes in human uterineleiomyoma cellsIn order to explore and extend our previous findings, we specificallysought to evaluate the effects of low (1μg/ml) and high (50μg/ml)concentrations of Gen on microarray gene expression patterns in UtLMcells. We performed a genome-wide analysis through the National Centerfor Toxicogenomics by using Agilent Whole Human Genome arrays.Using Ingenuity Pathway Analysis (IPA), we further analyzed thebiological significance of signature genes. Those genes werecharacterized into networks, functions and signaling pathways, whichwere extracted from the published scientific literature and stored in theIngenuity Pathways Knowledge Base (IPKB,http://www.ingeneuity.corn/products/Pathwaysknowledge.pdf). Real-timeRT-PCR and western blot analysis were used to further confirm specificgenes and pathways in high (50μg/ml) dose of Gen-treated UtLM cells.The main results are as follows:1. The molecular mechanisms of the stimulatory effect of low (1μg/ml)dose of Gen on UtLM cell: (1) Twenty-seven genes in UtLM cells treatedwith 1μg/ml of Gen with average differences of＞or = 1.5-fold weresignificantly expressed and regulated (P＜or = 0.001). (2) IPA revealedthat significant alterations in gene expression occurred in severalfunctions including cell-to-cell signaling and interaction, cellularmovement, cell morphology, cellular function and maintenance, cellulargrowth and proliferation and in a number of signaling pathways,involving cardiacβ-adrenergic, Wnt/β-catenin, TGF-βand inositol phosphate metabolism signaling. (3) Genes involved in those functionsand pathways, such as, up-regulation of A-myb, activin B, HOXA10,EDN1 and ID3, and down-regulation of MMP1, RGS2, PLN and PLAT,were identified for further investigation.2. The molecular mechanisms of the inhibitory effect of high (50μg/ml)dose of Gen on UtLM cell: (1) Five-hundred and forty-one genes inUtLM cells treated with 50μg/ml of Gen with average differences of＞or=1.5-fold were significantly expressed and regulated (P＜or = 0.001). (2)IPA revealed that significant alterations in gene expression occurred inseveral functions including cell cycle, cell death, cellular growth andproliferation and in a number of signaling pathways, involving integrin,VEGF, purine metabolism, TGF-βand fatty acid biosynthesis (Path 2). (3)Activin A, activin B, Smad3 and TGF-β2 gene expression levels were alldown-regulated in the TGF-βsignaling pathway. In addition, there wasdownregulation of genes related to cell cycle regulation, such as CDK6,A-myb, and Cyclin B2, while P15, a CDK inhibitor was upregulated. (4)Expression data for the above genes were identified and validated byreal-time reverse transcriptase-polymerase chain reaction studies. Geneand mRNA expression data for activin A and Smad3 were furtherconfirmed by western blot analysis of lysates of Gen treated UtLM cells.(5) In agreement with decreased gene, mRNA and protein expression ofactivin A in Gen treated UtLM cells, the growth of UtLM cells wasstimulated under activin A treatment and that simultaneous incubation ofUtLM cells with activin A partially abrogated the inhibitory effect ofgenistein. (6) The elevated expression of activin A and Smad3 in humanuterine leiomyoma tissue samples in comparison with autologousmyometrium further supported the contribution of activin A and Smad3in promoting the growth of UtLM cells. Conclusions: Specific genes, A-myb and activin B, were identified inboth stimulatory and inhibitory effect of low and high concentrations ofgenistein on UtLM cells, respectively. The inhibitory effect of highconcentration of Gen on UtLM cells may be through cell cycledysregulation and partially via down-regulation of activin A and Smad3through the TGF-βsignaling pathway.SectionⅣ: Low-dose genistein promotes the interactionsbetween estrogen receptor alpha and insulin-like growth factor-Ⅰreceptor leading to uterine leiomyoma cell proliferationLow doses of Genstimulate estrogen responsive human uterineleiomyoma (UtLM) "fibroid" cells, but not normal uterine smooth musclecells (UtSMC). In this part, we evaluated the roles of the estrogenreceptor (ER) and insulin-like growth factor-Ⅰreceptor (IGF-IR)pathways in the proliferation of UtLM cells compared to UtSMCfollowing genistein treatment at 1μg/ml. Using an estrogen responseelement (ERE) reporter assay and real-time RT-PCR, we found that Geninduced a higher transactivational potential and mRNA expression ofprogesterone receptor (PR) and insulin-like growth factor-Ⅰ(IGF-Ⅰ) inUtLM cells compared to UtSMC. Western blotting showed a decreasedexpression of estrogen receptor alpha (ERα), early activation ofextracellular regulated kinase (ERK) and Src homology/collagen (Shc) inUtLM cells, but not UtSMC. Additionally, in UtLM cells, Gen-inducedearly phosphorylation of ERK and the stimulatory effect were inhibitedby the estrogen antagonist, ICI 182,780, and MEK kinase inhibitor, PD98059, respectively. Immunoprecipitation data revealed that earlyinteractions between ERαand IGF-IR were ER-dependent in Gen-treatedUtLM cells.Conclusions: ERαis involved in the early events of the activation ofERK/MAPK and is preferentially activated in UtLM cells by a lowconcentration of Gen. Also, activation of ERK/MAPK signaling may beinvolved in the elevated ER transactivation observed in UtLM cells. Earlyinteractions between ER and IGF-IR signaling pathways may contributeto the hyper-responsiveness of UtLM cells compared to UtSMC after Gentreatment.