Uterine Cell Proliferation Research Articles

Abstract 60: Mig-6 suppresses development and progression of endometrial cancer by inhibiting ERK2 phosphorylation

Abstract Endometrial cancer is the most common cancer of the female reproductive system. PTEN is mutated or absent in more than half of human endometrial cancers. The major pathologic phenomenon of endometrial cancer is the loss of ovarian steroid hormone control over uterine epithelial cell proliferation and apoptosis. Mig-6 suppresses estrogen signaling. Here, we show the significance of MIG-6 in human endometrial cancer through sample analysis, where MIG-6 expression is inversely associated with ERK phosphorylation; this relationship is tightly correlated with endometrial cancer progression. To determine the tumor suppressor function of Mig-6 in the development of endometrial cancer, we generated Mig-6 conditional overexpression mice (R26Mig-6LSL). To assess the effects of Mig-6 on the PTEN/PI3K/AKT signaling pathway in uterine tumorigenesis, mice with Pten floxed (Ptenf/f) and R26Mig-6LSL were bred to the PRCre mouse model to generate overexpression of Mig-6 and ablation of Pten in the uterus (PRcre/+ R26Mig-6LSL Ptenf/f). PRcre/+ R26Mig-6LSL Ptenf/f showed significantly increased survival time and uterine weight compared to PRcre/+ Ptenf/f mice. Gross morphology and histological analysis displayed dramatically suppressed development of endometrial cancer in double mutant mice compared to ablation of Pten alone. Immunohistochemical analysis showed significantly increased apoptosis and decreased proliferation in epithelial cells of PRcre/+ R26Mig-6LSL Ptenf/f mice compared to PRcre/+ Ptenf/f mice. Interestingly, the expression of pERK1/2 was significantly decreased in PRcre/+ R26Mig-6LSL Ptenf/f mice compared to PRcre/+ Ptenf/f mice. To examine whether inhibition of ERK phosphorylation suppresses tumor progression in endometrial cancer, PRcre/+Ptenf/f mice were treated with U0126, an effective inhibitor of MAPK/ERK kinase. PRcre/+Ptenf/f mice treated with U0126 exhibited a significant reduction in uterine weight. Histopathological analysis of the entire animal cohort showed that inhibition of ERK phosphorylation suppressed endometrial cancer progression in PRcre/+ Ptenf/f mice, as reflected by the arrest of tumors at the hyperplastic or normal stage, whereas tumors from PRcre/+Ptenf/f mice treated with vehicle advanced to endometrial cancer. These results demonstrate that activation of ERK signaling is critical for endometrial cancer development and progression in Pten mutation. Our findings highlight a crucial tumor suppressor role for MIG-6 in progression of PTEN-null endometrial cancer by inhibiting ERK phosphorylation. As MIG-6 is a mediator of progesterone signaling, the activity of which can suppress unopposed-estrogen signaling, our studies provide a potential new drug target for the intervention of metastatic human endometrial cancer. (This work was supported by NIH, U54 HD007495 to J.P.L, NIH, R01 HD057873 and American Cancer Society Research Grant, RSG-12-084-01-TBG to J.W.J.) Citation Format: Jung-Yoon Yoo, Tae Hoon Kim, Hong Im Kim, Jane Li, Gordon B. Mills, Russell R. Broaddus, John P. Lydon, Ho-Geun Yoon, Jae-Wook Jeong. Mig-6 suppresses development and progression of endometrial cancer by inhibiting ERK2 phosphorylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 60. doi:10.1158/1538-7445.AM2014-60

Antitumor Effects of Flavopiridol on Human Uterine Leiomyoma In Vitro and in a Xenograft Model

Dysregulated cyclin-dependent kinases (CDKs) are considered a potential target for cancer therapy. Flavopiridol is a potent CDK inhibitor. In this study, the antiproliferative effect of the flavonoid compound flavopiridol and its mechanism in human uterine leiomyoma cells were investigated. The present study focused on the effect of flavopiridol in cell proliferation and cell cycle progression in primary cultured human uterine leiomyoma cells. Cell viability and cell proliferation assays were conducted. Flow cytometry was performed to determine the effect of flavopiridol on cell cycle. The expression of cell cycle regulatory-related proteins was evaluated by Western blotting. Cell viability and proliferation of uterine leiomyoma cells were significantly reduced by flavopiridol treatment in a dose-dependent manner. Flow cytometry results showed that flavopiridol induced G1 phase arrest. Flavopiridol-induced growth inhibition in uterine leiomyoma cells was associated with increased expression of p21(cip/wafl) and p27(kip1) in a dose-dependent manner. Downregulation of CDK2/4 and Cyclin A with a concomitant increase in dephosphorylation of retinoblastoma was observed. This study demonstrates that flavopiridol inhibits cell proliferation by initiating G1 cell cycle arrest in human uterine leiomyoma. We also found that flavopiridol is effective in inhibiting xenografted human uterine leiomyoma growth. These results indicate that flavopiridol could prove to be a promising chemopreventive and therapeutic agent for human uterine leiomyoma.

Uterine epithelial cell proliferation and endometrial hyperplasia: evidence from a mouse model.

In the uterus, epithelial cell proliferation changes during the estrous cycle and pregnancy. Uncontrolled epithelial cell proliferation results in implantation failure and/or cancer development. Transforming growth factor-β (TGF-β) signaling is a fundamental regulator of diverse biological processes and is indispensable for multiple reproductive functions. However, the in vivo role of TGF-β signaling in uterine epithelial cells remains poorly defined. We have shown that in the uterus, conditional deletion of the Type 1 receptor for TGF-β (Tgfbr1) using anti-Müllerian hormone receptor type 2 (Amhr2) Cre leads to myometrial defects. Here, we describe enhanced epithelial cell proliferation by immunostaining of Ki67 in the uteri of these mice. The aberration culminated in endometrial hyperplasia in aged females. To exclude the potential influence of ovarian steroid hormones, the proliferative status of uterine epithelial cells was assessed following ovariectomy. Increased uterine epithelial cell proliferation was also revealed in ovariectomized Tgfbr1 Amhr2-Cre conditional knockout mice. We further demonstrated that transcript levels for fibroblast growth factor 10 (Fgf10) were markedly up-regulated in Tgfbr1 Amhr2-Cre conditional knockout uteri. Consistently, treatment of primary uterine stromal cells with TGF-β1 significantly reduced Fgf10 mRNA expression. Thus, our findings suggest a potential involvement of TGFBR1-mediated signaling in the regulation of uterine epithelial cell proliferation, and provide genetic evidence supporting the role of uterine epithelial cell proliferation in the pathogenesis of endometrial hyperplasia.

Follistatin is essential for normal postnatal development and function of mouse oviduct and uterus

Female mice lacking the follistatin gene but expressing a human follistatin-315 transgene (tghFST315) have reproductive abnormalities (reduced follicles, no corpora lutea and ovarian-uterine inflammation). We hypothesised that the absence of follistatin-288 causes the abnormal reproductive tract via both developmental abnormalities and abnormal ovarian activity. We characterised the morphology of oviducts and uteri in wild type (WT), tghFST315 and follistatin-knockout mice expressing human follistatin-288 (tghFST288). The oviducts and uteri were examined in postnatal Day-0 and adult mice (WT and tghFST315 only) using histology and immunohistochemistry. Adult WT and tghFST315 mice were ovariectomised and treated with vehicle, oestradiol-17β (100ng injection, dissection 24h later) or progesterone (1mg×three daily injections, dissection 24h later). No differences were observed in the oviducts or uteri at birth, but abnormalities developed by adulthood. Oviducts of tghFST315 mice failed to coil, the myometrium was disorganised, endometrial gland number was reduced and oviducts and uteri contained abundant leukocytes. After ovariectomy, tghFST315 mice had altered uterine cell proliferation, and inflammation was maintained and exacerbated by oestrogen. These studies show that follistatin is crucial to postnatal oviductal-uterine development and function. Further studies differentiating the role of ovarian versus oviductal-uterine follistatin in reproductive tract function at different developmental stages are warranted.

Salinomycin inhibited cell proliferation and induced apoptosis in human uterine leiomyoma cells.

ObjectiveThe aim of this study was to investigate the anti-proliferative effect of the salinomycin in cell proliferation and apoptosis in primary cultured human uterine leiomyoma cells.MethodsCell viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Caspase-3 activity assay and DNA fragmentation assay were performed to determine the effect of apoptosis. The expression of apoptosis regulatory-related proteins was evaluated by western blot.ResultsThe cell viability and proliferation of uterine leiomyoma cells were significantly reduced by salinomycin treatment in a dose-dependent manner. DNA fragmentation assay results showed apoptotic cell death after salinomycin incubation. Salinomycin activated caspase-3, -8, and -9, causing apoptosis in uterine leiomyoma cells. Down-regulation of Bcl-2, XIAP, and FLIP with a concomitant increase in Bax, Fas, and DR5 were observed.ConclusionThese results provided the first evidence that salinomycin induce both intrinsic and extrinsic apoptosis. Therefore, salinomycin may be a promising chemopreventive and therapeutic agent against human uterine leiomyoma.

Carbon Monoxide Promotes Proliferation of Uterine Natural Killer Cells and Remodeling of Spiral Arteries in Pregnant Hypertensive Heme Oxygenase-1 Mutant Mice

Heme Oxygenase-1 (HO-1) and its metabolite carbon monoxide (CO) promote implantation and placentation. Pregnancy disorders such as preeclampsia and intrauterine growth restriction are linked to both HO-1 diminution and impaired remodeling of maternal spiral arteries (SAs). Here, we investigated whether CO is able to prevent preeclampsia and intrauterine growth restriction through the modulation of uterine natural killer (uNK) cells that are necessary for initiation of SA remodeling. Hmox1(+/-) or Hmox1(-/-) implantations presented fewer uNK cell numbers and lower expression of uNK-related angiogeneic factors compared with Hmox1(+/+) sites. Quantitative histology revealed that Hmox1(+/-) and Hmox1(-/-) implantations had shallow SA development that was accompanied by intrauterine growth restriction and gestational hypertension. Application of CO at low dose during early to midgestation prevented intrauterine growth restriction in Hmox1(+/-) mothers, this being associated with enhanced in situ proliferation of uNK cells and normalization of angiogenic parameters. Most importantly, CO improved SA remodeling and normalized blood pressure, ensuring a proper fetal growth. Thus, CO emerges as a key molecular player in pregnancy success by modulating uNK cells, which results in promotion of SA remodeling, adequate fetal support/growth, and prevention of hypertension.

17β-Estradiol alters oxidative stress response protein expression and oxidative damage in the uterus

The steroid hormone 17β-estradiol (E2) has profound effects on the uterus. However, with the E2-induced increase in uterine cell proliferation and metabolism comes increased production of reactive oxygen species (ROS). We examined the expression of an interactive network of oxidative stress response proteins including thioredoxin (Trx), Cu/Zn superoxide dismutase (SOD1), apurinic endonuclease (Ape1), and protein disulfide isomerase (PDI). We demonstrated that treatment of ovariectomized C57BL/6J female mice with E2 increased the mRNA and protein levels of Trx, but decreased SOD1 and Ape1 mRNA and protein expression. In contrast, E2 treatment increased PDI protein levels but had no effect on PDI transcript levels. Interestingly, E2 treatment also increased two markers of cellular damage, lipid peroxidation and protein carbonylation. Our studies suggest that the decreased expression of SOD1 and Ape1 caused by E2 treatment may in the long term result in disruption of ROS regulation and play a role in endometrial carcinogenesis.

The AF-1 activation function of estrogen receptor alpha is necessary and sufficient for uterine epithelial cell proliferation in vivo

The AF-1 activation function of estrogen receptor alpha is necessary and sufficient for uterine epithelial cell proliferation in vivo

Paricalcitol, vitamin D3 receptor agonist is superior to vitamin d3 in reducing proliferation of human uterine leiomyoma cells

Uterine leiomyomas are the most common benign tumors in black than in white women. Vitamin D deficiency is more prevalent among blacks (40-45%) compared to whites (4%). We and others have demonstrated that Vitamin D deficiency is a novel risk factor for the occurrence of human leiomyoma (Sabry et. al., 2013). We have also recently demonstrated that vitamin D treatment can shrink uterine leiomyoma lesion in the Eker rat model (Halder et. al., 2012).

Curcumin Induces Cross-Regulation Between Autophagy and Apoptosis in Uterine Leiomyosarcoma Cells

Uterine leiomyosarcoma (LMS) has an unfavorable response to standard chemotherapy. A natural occurring compound, curcumin, has been shown to have inhibitory effects on cancers. We previously demonstrated that curcumin reduced uterine LMS cell proliferation by targeting the AKT-mTOR pathway and activating apoptosis. To further explore the anticancer effect of curcumin, we investigated the efficacy of curcumin on autophagy in LMS cells. Cell proliferation in human uterine LMS cell lines, SKN and SK-UT-1, was assessed after exposure to rapamycin or curcumin. Autophagy was detected by Western blotting for light chain 3 and sequestosome 1 (SQSTM1/p62) expression. Apoptosis was confirmed by Western blotting for cleaved poly (ADP-ribose) polymerase (PARP). Both rapamycin and curcumin potently inhibited SKN and SK-UT-1 cell proliferation in a dose-dependent manner. Curcumin induced autophagy and apoptosis in SKN and SK-UT-1 cells, whereas rapamycin, a specific mTOR inhibitor, did not. Curcumin increased extracellular signal-regulated kinase 1/2 activity in both SKN and SK-UT-1 cells, whereas PD98059, an MEK1 inhibitor, inhibited both the extracellular signal-regulated kinase 1/2 pathway and curcumin-induced autophagy. These experimental findings suggest that curcumin is a potent inhibitor of cell proliferation in uterine LMS and provide new insights about ongoing signaling events leading to the possible development of a new therapeutic agent.

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

Agonistic activity of ICI 182 780 on activation of GSK 3β/AKT pathway in the rat uterus during the estrous cycle

We examined the ability of ICI 182,780 (ICI) to block uterine cell proliferation via protein kinase b/AKT pathway in the uterus of the rat during the estrous cycle. Intact rats, with regular estrous cycles, received a subcutaneous (s.c.) injection of either vehicle or ICI at 08:00h on the day of proestrus or at 00:00h on the day of estrus and sacrificed at 13:00h of metaestrus. Estradiol (E2) and progesterone (P4) plasma levels were measured by radioimmunoassay. Both ICI treatments, induced a significant decrease (p<0.01) in uterine estrogen receptor alpha (ERα) content, had no effect on uterine progesterone receptor (PR) protein expression and caused marked nuclear localization of cyclin D1, in both luminal and glandular uterine epithelium, as compared to vehicle-treated animals. Furthermore, we detected that ICI treatment induced glycogen synthase kinase (Gsk3-β) Ser 9 phosphorylation, which correlates with cyclin D1 nuclear localization. However, some differences were observed between the two different time schedules of administration. We observed that the administration of ICI at 08:00h on proestrus day produced a 15% inhibition of luminal epithelial cell proliferation, reduced uterine wet weight by 21% and caused reduction of Akt phosphorylation at Ser 473 as compared to vehicle-treated animals, whereas ICI treatment at 00:00h on estrus day had no effect on these parameters. The overall results indicate that ICI may exert agonistic and antagonistic effects on uterine cell proliferation through differential activation of the Akt pathway depending on the administration period during the estrous cycle, and indicates that the mechanism of cell proliferation during the physiological conditions of the estrous cycle, is under a different and more complex regulation than in the ovariectomized +E2 animal model.

Genetic Diversity in Normal Cell Populations is the Earliest Stage of Oncogenesis Leading to Intra-Tumor Heterogeneity

Random mutations and epigenetic alterations provide a rich substrate for microevolutionary phenomena to occur in proliferating epithelial tissues. Genetic diversity resulting from random mutations in normal cells is critically important for understanding the genetic basis of oncogenesis. However, evaluation of the cell-specific role of individual (epi-)genetic alterations in living tissues is extremely difficult from a direct experimental perspective. For this purpose, we have developed a single cell model to describe the fate of every cell in the uterine epithelium and to simulate occurrence of the first cancer cell. Computational simulations have shown that a baseline mutation rate of two mutations per cell division is sufficient to explain sporadic endometrial cancer as a rare evolutionary consequence with an incidence similar to that reported in SEER data. Simulation of the entire oncogenic process has allowed us to analyze the features of the tumor-initiating cells and their clonal expansion. Analysis of the malignant features of individual cancer cells, such as de-differentiation status, proliferation potential, and immortalization status, permits a mathematical characterization of malignancy at the single cell level and a comparison of intra-tumor heterogeneity between individual tumors. We found, under the conditions specified, that cancer stem cells account for approximately 7% of the total cancer cell population. Therefore, our mathematical modeling describes the genetic diversity and evolution in a normal cell population at the early stages of oncogenesis and characterizes intra-tumor heterogeneity. This model has explored the role of accumulation of a large number of genetic alterations in oncogenesis as an alternative to traditional biological approaches emphasizing the driving role of a small number of genetic mutations. A quantitative description of the contribution of a large set of genetic alterations will allow the investigation of the impact of environmental factors on the growth advantage of and selection pressure on individual cancer cells for tumor progression.

Mechanism of human chorionic gonadotrophin-mediated immunomodulation in pregnancy

Human chorionic gonadotrophin (hCG) is released within hours of fertilization and has a profound ability to downregulate maternal cellular immunity against trophoblastic paternal antigens. It also promotes angiogenic activity of the extravillous trophoblast, and impairment of this function may lead to inadequate placentation and an increased risk of preeclampsia. There is increasing evidence that hCG alters the activity of dendritic cells via an upregulation of indoleamine 2,3-dioxygenase activity. This reduces T-cell activation and cytokine production, as well as encouraging Treg cell recruitment to the fetal–maternal interface. These changes are critical in promoting maternal tolerance. hCG is also able to increase the proliferation of uterine natural killer cells, while reducing the activity of cytotoxic peripheral blood natural killer cells. There are rare reports of autoantibodies directed against hCG or the luteinizing hormone/hCG receptor in women with recurrent miscarriage. These autoantibodies are more frequent in women with thyroid autoimmunity. This may explain the association between thyroid autoimmunity and impaired fertility. Downregulating these anti-hCG and anti-luteinizing hormone/hCG receptor autoantibodies may be helpful in some women with early miscarriage or recurrent failed in vitro fertilization.

Overexpression of Cyclin D3 Improves Decidualization Defects in Hoxa-10−/− Mice

Uterine decidualization, a crucial process for implantation, is a tightly regulated process encompassing proliferation, differentiation, and polyploidization of uterine stromal cells. Hoxa (Homeobox A)-10, a homeobox transcription factor, is highly expressed in decidualizing stromal cells. Targeted gene deletion experiments have demonstrated marked infertility resulting from severely compromised decidualization in Hoxa-10(-/-) mice. However, the underlying mechanism by which Hoxa-10 regulates stromal cell differentiation remains poorly understood. Cyclin D3, a G(1) phase cell-cycle regulatory protein involved in stromal cell proliferation and decidualization, is significantly reduced in Hoxa-10(-/-) mice. The expression of cyclin D3 in the pregnant mouse uterus parallels stromal cell decidualization. Here, we show that adenovirus-driven cyclin D3 replacement in Hoxa-10(-/-) mice improves stromal cell decidualization. To address our question of whether cyclin D3 replacement in Hoxa-10(-/-) mice can improve decidualization, both in vitro and in vivo studies were completed after the addition of cyclin D3 or empty (control) viral vectors. Immunostaining demonstrated increased proliferation and decidualization in both in vitro and in vivo studies, and in situ hybridization confirmed increased expression of decidualization markers in vivo. Placentation was demonstrated as well in vivo in the cyclin D3-replaced animals. However, fertility was not restored in Hoxa-10(-/-) mice after d 10 of pregnancy. Finally, we identified several downstream targets of cyclin D3 during decidualization in vitro via proteomics experiments, and these were confirmed using in situ hybridization in vivo. Collectively, these results demonstrate that cyclin D3 expression influences a host of genes involved in decidualization and can improve decidualization in Hoxa-10(-/-) mice.

Impacts of linseed meal and estradiol-17β on cellularity, angiogenic and vasoactive factor mRNA expression, and vascularity of the uterus in ovariectomized ewes

Ilse, B. R., O'Neil, M. R., Lardy, G. P., Reynolds, L. P. and Vonnahme, K. A. 2012. Impacts of linseed meal and estradiol-17β on cellularity, angiogenic and vasoactive factor mRNA expression, and vascularity of the uterus in ovariectomized ewes. Can. J. Anim. Sci. 92: 297–306. The objective of the current study was to determine the estrogenic potential of the phytoestrogen secoisolariciresinol diglycoside (SDG) found in linseed meal (LSM) on uterine cell proliferation, vascularity, and angiogenic factor mRNA expression. Ovariectomized ewes (n=48) were fed a diet containing 12.5% LSM for 0, 1, 7, or 14 d and implanted with estradiol-17β (E2) for 0, 6, or 24 h before tissue collection. There was an interaction of LSM×E2 on uterine mass (grams; P=0.03; percentage change; P&lt;0.003). Uterine mass increased (P≤0.02) after 24 h of E2 exposure on days 1, 7, and 14 of LSM feeding, with the greatest mass occurring in ewes exposed to E2 for 24 h and 1 d LSM feeding. Regardless of days fed LSM, after 24 h of E2 exposure uterine mass was greatest. The greatest percentage increase in uterine mass occurred in ewes exposed to E2 for 24 h and fed 1 d of LSM. Cell proliferation within the uterine luminal epithelium was greatest (P&lt;0.01) with 24 h of E2 exposure compared with 0 h and 6 h. When expressed as the percentage change in uterine cell proliferation, feeding LSM for 14 d negated these effects. Only length of E2 exposure impacted vascularity with capillary number density at 6 h of E2 exposure being greater (P=0.02) than at 24 h. While mRNA expression of several angiogenic factors was influenced by E2, there was a LSM×E2 interaction (P≤0.03) only on vascular endothelial growth factor receptor 2 and fibroblast growth factor receptor 2C. It appears that growth and angiogenesis of E2 sensitive tissues may be influenced by the duration of LSM feeding.

The antidiabetic drug metformin inhibits uterine leiomyoma cell proliferation via an AMP-activated protein kinase signaling pathway

Uterine leiomyomas are the most common gynecological benign tumors and greatly affect reproductive health and wellbeing. Metformin is the most widely used antidiabetic drug in the world, and there is increasing evidence of a potential efficacy of this agent as an anticancer drug. In order to understand metformin’s anti-tumorigenic potential better, in this study, we investigated the inhibitory effect of metformin and expression of key targets of metformin cell signaling in leiomyoma cells. Cell proliferation was assessed after exposure to metformin. Apoptosis was assessed by western blotting for cleaved-PARP and TUNEL staining. The expressions of phosphorylated AMPK and phosphorylated S6 were determined by western blotting. Metformin potently inhibited ELT-3 cell proliferation in a dose-dependent manner. Western blotting analysis demonstrated that metformin induced phosphorylation of AMPK and the inhibitory effect was attenuated with AMPK inhibitor, compound C. In parallel, treatment with metformin decreased phosphorylation of S6 protein. These experimental findings show that metformin is a potent inhibitor of cell proliferation in leiomyoma cells. This effect is mediated by AMPK activation and subsequent inhibition of the mTOR pathway. Thus, this study provides a possible mechanism of the action of metformin in the inhibition of leiomyoma cell growth.

Dysregulation of Uterine Signaling Pathways in Progesterone Receptor-Cre Knockout of Dicer

Epithelial-stromal interactions in the uterus are required for normal uterine functions such as pregnancy, and multiple signaling pathways are essential for this process. Although Dicer and microRNA (miRNA) have been implicated in several reproductive processes, the specific roles of Dicer and miRNA in uterine development are not known. To address the roles of miRNA in the regulation of key uterine pathways, we generated a conditional knockout of Dicer in the postnatal uterine epithelium and stroma using progesterone receptor-Cre. These Dicer conditional knockout females are sterile with small uteri, which demonstrate significant defects, including absence of glandular epithelium and enhanced stromal apoptosis, beginning at approximately postnatal d 15, with coincident expression of Cre and deletion of Dicer. Specific miRNA (miR-181c, -200b, -101, let-7d) were down-regulated and corresponding predicted proapoptotic target genes (Bcl2l11, Aldh1a3) were up-regulated, reflecting the apoptotic phenomenon. Although these mice had normal serum hormone levels, critical uterine signaling pathways, including progesterone-responsive genes, Indian hedgehog signaling, and the Wnt/β-catenin canonical pathway, were dysregulated at the mRNA level. Importantly, uterine stromal cell proliferation in response to progesterone was absent, whereas uterine epithelial cell proliferation in response to estradiol was maintained in adult uteri. These data implicate Dicer and appropriate miRNA expression as essential players in the regulation of multiple uterine signaling pathways required for uterine development and appropriate function.

Progesterone and DNA Damage Encourage Uterine Cell Proliferation and Decidualization through Up-regulating Ribonucleotide Reductase 2 Expression during Early Pregnancy in Mice

Embryo implantation into the maternal uterus is a crucial step for the successful establishment of mammalian pregnancy. Following the attachment of embryo to the uterine luminal epithelium, uterine stromal cells undergo steroid hormone-dependent decidualization, which is characterized by stromal cell proliferation and differentiation. The mechanisms underlying steroid hormone-induced stromal cell proliferation and differentiation during decidualization are still poorly understood. Ribonucleotide reductase, consisting of two subunits (RRM1 and RRM2), is a rate-limiting enzyme in deoxynucleotide production for DNA synthesis and plays an important role in cell proliferation and tumorgenicity. Based on our microarray analysis, Rrm2 expression was significantly higher at implantation sites compared with interimplantation sites in mouse uterus. However, the expression, regulation, and function of RRM2 in mouse uterus during embryo implantation and decidualization are still unknown. Here we show that although both RRM1 and RRM2 expression are markedly induced in mouse uterine stromal cells undergoing decidualization, only RRM2 is regulated by progesterone, a key regulator of decidualization. Further studies showed that the induction of progesterone on RRM2 expression in stromal cells is mediated by the AKT/c-MYC pathway. RRM2 can also be induced by replication stress and DNA damage during decidualization through the ATR/ATM-CHK1-E2F1 pathway. The weight of implantation sites and deciduoma was effectively reduced by specific inhibitors for RRM2. The expression of decidual/trophoblast prolactin-related protein (Dtprp), a reliable marker for decidualization in mice, was significantly reduced in deciduoma and steroid-induced decidual cells after HU treatment. Therefore, RRM2 may be an important effector of progesterone signaling to induce cell proliferation and decidualization in mouse uterus.

Gestrinone inhibits growth of human uterine leiomyoma may relate to activity regulation of ERα, Src and P38 MAPK

The study was to investigate the effect of gestrinone on the growth of human uterine leiomyoma cells and on the levels and activity of p38, Src and estrogen receptor alpha (ERα). Human uterine leiomyoma cells were cultured and treated with dimethylsulfoxide (DMSO) or a gestrinone concentration gradient. Morphological changes were observed and apoptosis was evaluated. Levels of p38 and phosphorylated-p38 (pp38) were assayed by enzyme-linked immunosorbent assay (ELISA). Levels of ERα and Src were analyzed using real-time RT-PCR and Western blotting. The result showed that gestrinone significantly inhibited the growth of cultured human uterine leiomyoma cells in a concentration- and time-dependent manner, with a 50% inhibitory concentration (IC50) value and corresponding 95% confidence intervals (CI) of 43.67 (23.46∼81.32), 27.78 (12.51∼61.68) and 15.25 (7.17∼32.43) μmol/L at 20, 40 and 60h, respectively. Compared with control-treated leiomyoma cells, gestrinone significantly reduced both the expression of ERα (P<0.05) and the levels of phospho-Ser167-ERα (P<0.05). Gestrinone also markedly suppressed the level of phospho-Tyr416-Src (P<0.05). Moreover, gestrinone significantly increased the ratio of phospho-p38/p38 mitogen-activated protein kinase (MAPK) (P<0.05). However, no significant increase in apoptosis or cell cycle arrest was observed (P>0.05) in response to the tested concentrations of 0.1 to 3.0μmol/L. As a conclusion, gestrinone suppresses the proliferation of uterine leiomyoma cells mainly by regulating the activity of ERα/Src/p38 MAPK in a concentration-dependent manner at a low concentration of 0.1∼3.0μM, but not significantly regulating apoptosis. Gestrinone opposes the growth of uterine leiomyoma through multiple genes.