Ayman Al-Hendy

Catechol-O-Methyltransferase Polymorphism Is Associated With Increased Uterine Leiomyoma Risk in Different Ethnic Groups

Objectives: Uterine leiomyomas (ULMs) are estrogen-dependent tumors that are more common in African American women. The etiology for such ethnic disparity is currently unknown. catechol-O-mythyltransferase (COMT) is an essential enzyme in estrogen metabolism. In the current study, we investigated the association of the functional COMT Val158 Met polymorphism with ULM in different ethnic groups. We also studied the biologic role of COMT in tumor formation in human and rat leiomyoma cell lines and the potential therapeutic utility of COMT inhibitors. Methods: The genotype frequencies of the functional COMT Val158 Met polymorphism among participants with (186 women) or without (142 women) ULMs were compared, as was the differential ethnic distribution of that polymorphism using polymerase chain reaction (PCR) and restriction-fragment linkage polymorphism. Poliferation, Western blot, and reporter transactivation analyses were applied to myometrial and leiomyoma cells representative of different COMT genotypes. Results: Women with the high-activity COMT Val/Val genotype are 2.5 times more likely to develop ULMs than women with other genotypes (confidence interval, 1.017 to 6.151; P < .001). The prevalence of this genotype was significantly higher in African American women (47%) compared with white (19%) or Hispanic (30%) women (P = .003). Myometrial cell lines expressing the Val/Val genotype exhibited significantly enhanced responses to estrogen in proliferation and in estrogen-responsive element reporter assays. COMT-specific inhibitors reversed such a response and induced apoptosis. Myometrial specimens from Val/Val women demonstrated distinct estrogen-regulated gene expression that was consistent with enhanced proliferation and decreased apoptosis. Conclusions: The high-activity COMT Val/Val genotype is associated with increased risk of ULM. Our results provide a possible explanation for the higher prevalence of ULMs among African American women and offer a potential new target for nonsurgical treatment using COMT inhibitors.

1,25-Dihydroxyvitamin D3 Reduces TGF-β3-Induced Fibrosis-Related Gene Expression in Human Uterine Leiomyoma Cells

BACKGROUND Uterine leiomyomas (fibroids) are the most common benign estrogen-dependent tumors of premenopausal women. TGF-β3 up-regulates the synthesis of many of extracellular matrix proteins that are associated with tissue fibrosis. OBJECTIVE To examine the effect of 1,25-dihydroxyvitamin D(3) (vitamin D(3)) on TGF-β3-induced fibrosis-related protein expression in immortalized human uterine leiomyoma (HuLM) cells. METHODS HuLM cells were treated with TGF-β3 with or without vitamin D(3). Western blot analyses were employed to test the effect of vitamin D(3) on TGF-β3-induced protein expression of collagen type 1, fibronectin, and plasminogen activator inhibitor-1 proteins. Western blots as well as immunofluorescence analyses were used to verify the effect of vitamin D(3) on TGF-β3-induced Smad activation involved in extracellular matrix protein synthesis and deposition, which ultimately lead to tissue fibrosis. RESULTS We observed that TGF-β3 induced fibronectin and collagen type 1 protein expression in HuLM cells, and that effect was suppressed by vitamin D(3). TGF-β3 also induced protein expression of plasminogen activator inhibitor-1, an important TGF-β target, in HuLM cells, which was also inhibited by vitamin D(3). Additionally, TGF-β3 induced phosphorylation of Smad2 as well as nuclear translocation of Smad2 and Smad3 in HuLM cells, whereas vitamin D significantly reduced all these TGF-β3-mediated effects. Therefore, our results suggest that vitamin D(3) has consistently reduced TGF-β3 effects that are involved in the process of fibrosis in human leiomyoma cells. CONCLUSION Vitamin D(3) is an antifibrotic factor that might be potentially useful as a novel therapeutic for nonsurgical treatment of benign uterine fibroids.

Vitamin D inhibits proliferation of human uterine leiomyoma cells via catechol-O-methyltransferase.

OBJECTIVE To evaluate the effects and mechanisms of action of vitamin D on human uterine leiomyoma (HuLM) cell proliferation in vitro. DESIGN Laboratory study. SETTING University hospitals. PATIENTS(S) Not applicable. INTERVENTIONS(S) Not applicable. MAIN OUTCOME MEASURE(S) HuLM cells were treated with 1,25-dihydroxyvitamin D3 (vitamin D), and cell proliferation was assayed by the methylthiazolyl tetrazolium technique. proliferating cell nuclear antigen (PCNA), BCL-2, BCL-w, cyclin-dependent kinase (CDK) 1, and catechol-O-methyltransferase (COMT) protein levels were analyzed by Western blotting. COMT mRNA and enzyme activity were assayed by quantitative reverse-transcription polymerase chain reaction (RT-PCR) and high-performance liquid chromatography analysis, respectively. The role of COMT was evaluated in stable HuLM cells by silencing COMT expression. RESULT(S) Vitamin D inhibited the growth of HuLM cells by 47±0.03% at 1 μM and by 38±0.02% at 0.1 μM compared with control cells at 120 hours of treatment. Vitamin D inhibited extracellular signal-regulated kinase activation and down-regulated the expression of BCL-2, BCL-w, CDK1, and PCNA. Western blot, RT-PCR, and enzyme assay of COMT demonstrated inhibitory effects of vitamin D on COMT expression and enzyme activity. Silencing endogenous COMT expression abolished vitamin D-mediated inhibition of HuLM cell proliferation. CONCLUSION(S) Vitamin D inhibits growth of HuLM cells through the down-regulation of PCNA, CDK1, and BCL-2 and suppresses COMT expression and activity in HuLM cells. Thus, hypovitaminosis D appears to be a risk factor for uterine fibroids.

1,25-Dihydroxyvitamin D3 Treatment Shrinks Uterine Leiomyoma Tumors in the Eker Rat Model

ABSTRACT Uterine leiomyomas (fibroids) are the most common benign tumors in women of reproductive age. These tumors are three to four times more prevalent in African American women, who also have a 10 times higher incidence of hypovitaminosis D than white women. Recent studies have demonstrated the antitumor effects of 1,25-dihydroxyvitamin D3 on several cancers, but its effects on uterine leiomyomas are still unknown. To determine the antitumor and therapeutic effects of 1,25-dihydroxyvitamin D3 on uterine leiomyomas, female Eker rats (14–16 mo old) harboring uterine leiomyomas were randomized into control and experimental groups and were given vehicle versus 1,25-dihydroxyvitamin D3 (0.5 μg/kg per day) subcutaneously for 3 wk, respectively. At the end of the experiment, the rats were euthanized, and the leiomyoma tumors were analyzed. Treatment with 1,25-dihydroxyvitamin D3 significantly reduced leiomyoma tumor size in Eker rats. It also reduced leiomyoma size by suppressing cell growth and proliferation-related genes (Pcna, cyclin D1 [Ccnd1], Myc, Cdk1, Cdk2, and Cdk4), antiapoptotic genes (Bcl2 and Bcl2l1 [Bcl-x]), and estrogen and progesterone receptors. Additionally, immunohistochemistry revealed decreased expression of PCNA and MKI67 (a marker of proliferation) and increased expression of caspase 3 in 1,25-dihydroxyvitamin D3-treated Eker rat leiomyomas. Toxicity analyses using serum samples showed similar levels of SGOT, SGPT, calcium, and total bilirubin in 1,25-dihydroxyvitamin D3-treated and vehicle-treated control Eker rats. These results support that 1,25-dihydroxyvitamin D3 is an antitumor agent that may be a potential safe, nonsurgical therapeutic option for the treatment of uterine leiomyomas.

Vitamin D3 inhibits expression and activities of matrix metalloproteinase-2 and -9 in human uterine fibroid cells

STUDY QUESTION Can biologically active vitamin D3 [1,25(OH)₂D3] regulate the expression and activity of matrix metalloproteinases (MMPs) in human uterine fibroid cells? SUMMARY ANSWER 1,25(OH)₂D3 effectively reduced the expression and activities of MMP-2 and MMP-9 in cultured human uterine fibroid cells. WHAT IS KNOWN ALREADY Uterine fibroids (leiomyoma) express higher levels of MMP activity than adjacent normal myometrium, and this is associated with uterine fibroid pathogenesis. However, it is unknown whether 1,25(OH)₂D3 can regulate the expression and activities of MMPs in human uterine fibroid cells. STUDY DESIGN, SIZE, DURATION Surgically removed fresh fibroid tissue was used to generate primary uterine fibroid cells. PARTICIPANTS/MATERIALS, SETTING, METHODS An immortalized human uterine fibroid cell line (HuLM) and/or primary human uterine fibroid cells isolated from fresh fibroid tissue were used to examine the expression of several MMPs, tissue inhibitors of metalloproteinases (TIMP) 1 and 2 and the activities of MMP-2 and MMP-9 after 1,25(OH)₂D3 treatment. Real-time PCR and western blots analyses were used to measure mRNA and protein expression of MMPs, respectively. Supernatant cell culture media were analyzed for MMP-2 and MMP-9 activities using a gelatin zymography assay. MAIN RESULTS AND THE ROLE OF CHANCE 1-1000 nM 1,25(OH)₂D3 significantly reduced mRNA levels of MMP-2 and MMP-9 in HuLM cells in a concentration-dependent manner (P < 0.5 to P < 0.001). The mRNA levels of MMP-1, MMP-3, MMP-13 and MMP-14 in HuLM cells were also reduced by 1,25(OH)₂D3. 1,25(OH)₂D3 significantly reduced MMP-2 and MMP-9 protein levels in a concentration-dependent manner in both HuLM and primary uterine fibroid cells (P < 0.05 to P < 0.001). Moreover, 1,25(OH)₂D3 increased the mRNA levels of vitamin D receptor (VDR) and TIMP-2 in a concentration-dependent manner in HuLM cells (P < 0.05 to P < 0.01). 1,25(OH)₂D3 also significantly increased protein levels of VDR and TIMP-2 in all cell types tested (P < 0.05 to P < 0.001). Gelatin zymography revealed that pro-MMP-2, active MMP-2 and pro-MMP-9 were down-regulated by 1,25(OH)₂D3 in a concentration-dependent manner; however, the active MMP-9 was undetectable. LIMITATIONS, REASONS FOR CAUTION This study was performed using in vitro uterine fibroid cell cultures and the results were extrapolated to in vivo situation of uterine fibroids. Moreover, in this study the interaction of vitamin D3 with other regulators such as steroid hormone receptors was not explored. WIDER IMPLICATIONS OF THE FINDINGS This study reveals an important biological function of 1,25(OH)₂D3 in the regulation of expression and activities of MMP-2 and MMP-9. Thus, 1,25(OH)₂D3 might be a potential effective, safe non-surgical treatment option for human uterine fibroids.

Molecular genetics and racial disparities of uterine leiomyomas

Uterine leiomyomas (ULMs) are benign oestrogen-dependent tumours of the myometrium. They are the most common tumours of the female genital tract, affecting around 77% of the female population. ULMs are more common in Black women than White women. These tumours tend to develop earlier and be more numerous, larger in size and more symptomatic in Black women than other ethnic groups. The molecular mechanism underlying this ethnic disparity is not fully understood. Polymorphism of genes involved in oestrogen synthesis and/or metabolism (COMT, CYP17), variation in the expression levels or function of oestrogen and progesterone receptors or retinoic acid nuclear receptors (retinoid acid receptor-alpha, retinoid X receptor-alpha), or aberrant expression of micro-RNAs are some of the molecular mechanisms that may be involved.

Green tea extract inhibits proliferation of uterine leiomyoma cells in vitro and in nude mice.

OBJECTIVE The purpose of this study was to investigate the effect of epigallocatechin gallate (EGCG) on rat leiomyoma (ELT3) cells in vitro and in a nude mice model. STUDY DESIGN ELT3 cells were treated with various concentrations of EGCG. Cell proliferation, proliferation cell nuclear antigen (PCNA), and cyclin-dependent kinase 4 (Cdk4) protein levels were evaluated. ELT3 cells were inoculated subcutaneously in female athymic nude mice. Animals were fed 1.25 mg EGCG (in drinking water)/mouse/day. Tumors were collected and evaluated at 4 and 8 weeks after the treatment. RESULTS Inhibitory effect of EGCG (200 micromol/L) on ELT3 cells was observed after 24 hours of treatment (P < .05). At > or = 50 micromol/L, EGCG significantly decreased PCNA and Cdk4 protein levels (P < .05). In vivo, EGCG treatment dramatically reduced the volume and weight of tumors at 4 and 8 weeks after the treatment (P < .05). The PCNA and Cdk4 protein levels were significantly reduced in the EGCG-treated group (P < .05). CONCLUSION EGCG effectively inhibits proliferation and induces apoptosis in rat ELT3 uterine leiomyoma cells in vitro and in vivo.

FSH Receptor (FSHR) Expression in Human Extragonadal Reproductive Tissues and the Developing Placenta, and the Impact of Its Deletion on Pregnancy in Mice

ABSTRACT Expression and function of the follicle-stimulating hormone receptor (FSHR) in females were long thought to be limited to the ovary. Here, however, we identify extragonadal FSHR in both the human female reproductive tract and the placenta, and test its physiological relevance in mice. We show that in nonpregnant women FSHR is present on: endothelial cells of blood vessels in the endometrium, myometrium, and cervix; endometrial glands of the proliferative and secretory endometrium; cervical glands and the cervical stroma; and (at low levels) stromal cells and muscle fibers of the myometrium. In pregnant women, placental FSHR was detected as early as 8–10 wk of gestation and continued through term. It was expressed on: endothelial cells in fetal portions of the placenta and the umbilical cord; epithelial cells of the amnion; decidualized cells surrounding the maternal arteries in the maternal decidua; and the stromal cells and muscle fibers of the myometrium, with particularly strong expression at term. These findings suggest that FSHR expression is upregulated during decidualization and upregulated in myometrium as a function of pregnancy. The presence of FSHR in the placental vasculature suggests a role in placental angiogenesis. Analysis of genetically modified mice in which Fshr is lacking in fetal portions of the placenta revealed adverse effects on fetoplacental development. Our data further demonstrate FSHB and CGA mRNAs in placenta and uterus, consistent with potential local sources of FSH. Collectively, our data suggest heretofore unappreciated roles of extragonadal FSHR in female reproductive physiology.

Medical Treatment of Uterine Leiomyoma

Uterine leiomyomas (also called myomata or fibroids) are the most common gynecologic tumors in the United States. The prevalence of leiomyomas is at least 3 to 4 times higher among African American women than in white women. Pathologically, uterine leiomyomas are benign tumors that arise in any part of the uterus under the influence of local growth factors and sex hormones, such as estrogen and progesterone. These common tumors cause significant morbidity for women and they are considered to be the most common indication for hysterectomy in the world; they are also associated with a substantial economic impact on health care systems that amounts to approximately

Serum vitamin D3 level inversely correlates with uterine fibroid volume in different ethnic groups: a cross-sectional observational study.

2.2 billion/year in the United States alone. Uterine myomas cause several reproductive problems such as heavy or abnormal uterine bleeding, pelvic pressure, infertility, and several obstetrical complications including miscarriage and preterm labor. Surgery has traditionally been the gold standard for the treatment of uterine leiomyomas and has typically consisted of either hysterectomy or myomectomy. In recent years, a few clinical trials have evaluated the efficacy of orally administered medications for the management of leiomyoma-related symptoms. In the present review, we will discuss these promising medical treatments in further detail.