Proliferation Of Leiomyoma Cells Research Articles

Activin A induces leiomyoma cell proliferation, extracellular matrix (ECM) accumulation and myofibroblastic transformation of myometrial cells via p38 MAPK

ObjectiveThe objective of this study was to evaluate the role of Activin A and p38β MAPK-activated signaling in human leiomyoma cells, myometrial cells and mouse myometrial tissues. MethodsThe immortalization human leiomyoma cells (HuLM), the immortalized human myometrial cells (HM) and mouse myometrial tissues were treated with Activin A (4 nM) and/or specific p38 inhibitor SB202190 (10 μM) for different days of interval (to measure proliferation rate) or 1 h (to measure signaling molecules) or 48 h (to measure proliferating markers Ki-67, ECM mRNA, and/or ECM protein expression) by real-time PCR, Western blot, and/or immunocytochemistry. ResultsActivin A induced cell proliferation and ECM proteins accumulation in HuLM cells via p38 MAPK. Activin A also induced myofibroblastic transformation in HM cells and mouse myometrical tissues via the phosphorylation of p38. The effects of Activin A in leiomyoma cells, myometrical cells and tissues were abolished by p38α/β MAPK inhibitor SB202190. ConclusionThis study demonstrates Activin A-p38 MAPK signaling pathway in leiomyoma and myometrium may contribute to excessive ECM production, leiomyoma growth and progression. Targeting Activin A-p38 MAPK signaling pathway could be a potential therapeutic intervention for uterine leiomyoma.

Ulipristal acetate induces cell cycle delay and remodeling of extracellular matrix.

Uterine leiomyoma is a benign tumor that grows within the muscle tissue of the uterus. Ulipristal acetate (UPA) is a pre-operative drug used to reduce the size of leiomyoma. The aim of the present study was to examine the in vitro mechanistic details of action of UPA on uterine leiomyomas. Primary cultures of leiomyoma cells were isolated from patient myomectomy specimens and incubated in the presence or absence of UPA at various concentrations. The proliferation, cell viability and doubling time properties of the treated cells were analyzed. In addition, the mRNA and protein expression levels of p21, p27, cyclin E, cyclin-dependent kinase 2 (CDK2), matrix metalloproteinase (MMP)-2 and MMP-9 were examined, as well as the structure of F-actin in the primary-cultured leiomyoma cells. The results demonstrated that UPA exerted inhibitory effects on proliferation of primary-cultured leiomyoma cells. Expression of p21 and p27 was upregulated, while cyclin E and CDK2 were downregulated in UPA-treated primary-cultured leiomyoma cells. An increased expression of MMP-2 was observed in primary-cultured leiomyoma cells and a leiomyoma tissue sample of a patient with previous history of UPA treatment. Furthermore, a pronounced formation of F-actin stress fibers was observed in leiomyoma cells of the UPA-treated patient. These data suggest that UPA treatment attenuated the proliferation of uterine fibroid cells via upregulation of p21 and p27, resulting in cell cycle delay. The findings in the current study also suggest that UPA may cause extracellular matrix constriction, leading to the shrinkage in size of the leiomyoma possibly via stimulation of MMP-2 expression and induction of actin stress fibers.

Resveratrol inhibits human leiomyoma cell proliferation via crosstalk between integrin αvβ3 and IGF-1R

Leiomyomas (myomas) are the most common benign smooth muscle cell tumor of the myometrium. Resveratrol, a stilbene, has been used as an anti-inflammatory and antitumor agent. In the current study, we investigated the inhibitory effect of resveratrol on the proliferation of primary human myoma cell cultures. Resveratrol arrested cell proliferation via integrin αvβ3. It also inhibited integrin αvβ3 expression and protein accumulation. Concurrently, constitutive AKT phosphorylation in myoma cells was inhibited by resveratrol. Expressions of proapoptotic genes, such as cyclooxygenase (COX)-2, p21 and CDKN2, were induced by resveratrol in myoma cells. On the other hand, expressions of proliferative (anti-apoptotic) genes were either inhibited, as in BCL2, or unchanged, as in cyclin D1 and proliferating cell nuclear antigen (PCNA). The accumulation of insulin-like growth factor (IGF)-1 receptor (IGF-1R) was inhibited by resveratrol in primary myoma cells. IGF-1-induced cell proliferation was inhibited by co-incubation with resveratrol. Therefore, growth modulation of myoma cells occurs via mechanisms dependent on cross-talk between integrin αvβ3 and IGF-1R. Our findings suggest that resveratrol can be considered an alternative therapeutic agent for myomas.

Dysregulated chaperones associated with cell proliferation and negative apoptosis regulation in the uterine leiomyoma.

Uterine leiomyomas are benign smooth muscle cell tumors that originate from the myometrium. In this study we focus on dysregulated chaperones associated with cell proliferation and apoptosis. Paired tissue samples of 15 leiomyomas and adjacent myometria were obtained and analyzed by two-dimensional gel electrophoresis (2-DE). Mass spectrometry was used for protein identification and western blotting for 2-DE data validation. The values of 6 chaperones were found to be significantly different in the leiomyoma when compared with the myometrium. A total of 4 proteins were upregulated in the leiomyoma and 2 proteins were downregulated. Calreticulin and 78 kDa glucose-regulated protein were further validated by western blotting because the first is considered a marker of cell proliferation, while the second protects against apoptotic cell death. In addition, we also validated the two downregulated proteins heat shock protein β-1 and heat shock 70 kDa protein 1A. Our study shows the existence of a dysregulation of chaperone proteins associated with leiomyoma development. Functional studies are needed to ascertain the role of these chaperones in the leiomyoma. This may be crucial for the further development of specific inhibitors against the activity of these proteins in order to block the growth of the leiomyoma.

RANKL/RANK Pathway and Its Inhibitor RANK-Fc in Uterine Leiomyoma Growth.

Uterine leiomyomas are the most common type of gynecologic tumor in women. To determine the role of the cytokine receptor activator of nuclear factor κ-Β ligand (RANKL); its receptor, receptor activator of nuclear factor κ-Β (RANK); and the RANKL/RANK pathway inhibitor RANK-Fc in leiomyoma growth. Messenger RNA (mRNA) or protein levels of RANKL, RANK, and proliferation markers cyclin D1 and Ki67 were assessed in various leiomyoma tissues and cell populations. Human xenograft experiments were performed to determine the effects of RANK-Fc on leiomyoma growth in vivo. Research laboratory. Twenty-four regularly cycling premenopausal women (age 28 to 49 years) who were not receiving hormone therapy. None. Tumor growth in a murine xenograft model following targeting of the RANKL/RANK pathway with RANK-Fc. RANKL mRNA levels in leiomyoma were significantly higher than those in myometrial tissues. The highest RANK levels were found in the leiomyoma stem cell population, which is deficient in progesterone receptor (PR). Conversely, the highest RANKL levels were found in the PR-rich leiomyoma intermediate cell (LIC) population. R5020, a PR agonist, specifically increased RANKL expression in LICs. RANK-Fc blocked RANKL-induced expression of the proliferative gene cyclin D1. Treatment with RANK-Fc also significantly decreased tumor growth in vivo and diminished the expression of proliferation marker Ki67 in tumors (P < 0.01; n = 4). Treatment with the RANKL/RANK pathway inhibitor RANK-Fc significantly decreased human leiomyoma cell proliferation and tumor growth. This suggests that the RANKL/RANK pathway could serve as a potential target for the prevention and treatment of uterine leiomyoma.

Fucoidan Inhibits the Proliferation of Leiomyoma Cells and Decreases Extracellular Matrix-Associated Protein Expression

Background/Aims: Uterine leiomyomas (ULs) are benign uterine tumors, and the most notable pathophysiologic feature of ULs is excessive accumulation of extracellular matrix (ECM). Fucoidan is a polysaccharide extracted from brown seaweeds that has a wide range of pharmacological properties, including anti-fibrotic effects. We aimed to study the effect of fucoidan on the growth of ULs activated by transforming growth factor beta (TGFβ). Methods: We used ELT-3 (Eker rat leiomyoma tumor-derived cells) and HUtSMC (human uterine smooth muscle cells) as in vitro models. Cell viability was determined by the MTT assay. Cell colony formation was stained using crystal violet. The side population, cell cycle and apoptosis were analyzed using flow cytometry. Protein expression was assayed by western blot analysis. We also conducted in vivo experiments to confirm the inhibitory effects of fucoidan in nude mouse xenograft models. Tumor tissues were assayed by immunohistochemistry analysis. Results: In our study, fucoidan caused a 50% growth inhibition using a dose of 0.5 mg/ml and decreased the stem cell activity after 48 h. In addition, fucoidan induced sub-G1 cell cycle arrest and apoptosis. Fucoidan down-regulated fibronectin, vimentin, α-SMA and the COL1A1 protein levels in TGFβ3-induced ELT-3 cells. In the cellular mechanism, fucoidan abrogated TGFβ3-induced levels of p-Smad2 and p-ERK1/2, as well as β-catenin translocation into the nucleus. Furthermore, fucoidan suppressed xenograft tumor growth in vivo. Conclusion: Fucoidan displays anti-proliferation and anti-fibrotic effects and exerts protective effects against ULs development.

Ulipristal acetate regulates perilipin-2 gene expression through progesterone receptor isoform B in uterine leiomyoma cells

Progesterone and its receptor (PR) exert a tumorigenic effect on leiomyoma cells. There are two functionally distinct isoforms of PR: PRA and PRB. Ulipristal acetate (UPA), a selective PR modulator, has been clinically shown to reduce the size of leiomyomas, although the exact mechanisms are unknown. Using an unbiased ChIP-sequencing technique, we previously uncovered perilipin-2 (PLIN2) as a novel PR target gene in primary human uterine leiomyoma cells. PLIN2, an adipose differentiation-related protein, is a cytoplasmic lipid droplet binding protein that plays an important role in lipid metabolism. PLIN2 has been shown to mediate cell proliferation, apoptosis, and fibrogenic gene expression in various cell types such as pancreatic and hepatic stellate cells; however, little is known about the role of PLIN2 in the regulation of the growth of human uterine leiomyomas. The present study aimed to test whether UPA regulates PLIN2 expression and to determine the function of PLIN2 in leiomyoma cells. Basic science. Myometrial and leiomyoma tissues were collected from premenopausal women undergoing hysterectomies. We examined the impact of UPA treatment on PLIN2 gene expression and its function in leiomyoma cells by depleting PLIN2 and PR expression via small interfering RNA (siRNA). We also characterized the PLIN2 gene expression patterns between leiomyoma and matched myometrial tissues. We found that 75% of subjects’ leiomyoma tissue expressed lower mRNA levels of PLIN2 compared to the adjacent myometrial tissue. We also found that UPA treatment induced PLIN2 expression in a time and dose-dependent manner in primary leiomyoma cells. When we knocked down PR to determine whether ulipristal treatment induced PLIN2 expression through PR, we found that PR siRNA dramatically decreased the total PR level to 20%; however, it only mildly decreased PRB expression (80% of control level), suggesting that PR siRNA may disproportionately deplete endogenous PRA more efficiently than PRB. Intriguingly, UPA treatment consistently stimulated PLIN2 expression in the control (scrambled) siRNA group. PLIN2 expression was further increased in the PR siRNA group, suggesting that UPA upregulated PLIN2 expression predominantly through PRB and that PRA may function to inhibit PRB-mediated PLIN2 transcriptional activity. Furthermore, knockdown of PLIN2 with siRNA significantly increased protein levels of proliferating cell nuclear antigen (a marker for cell proliferation). We present evidence that UPA stimulates PLIN2 expression and PLIN2 represses cell proliferation in primary uterine leiomyoma. Our findings also suggest that PRA and PRB may play differential roles in leiomyoma growth. We speculate that PLIN2 plays an important role in mediating the therapeutic effects of ulipristal in uterine leiomyomas.

Molecular mechanisms of regulation of uterine leiomyoma growth

Background: to elucidate the molecular mechanisms, regulating growth and proliferation of leiomyoma cells, the estimation of the activity of PR-A and PR-B isoforms of progesterone receptor and PI3K/Akt and MAPК kinases, participating in signal transduction from sex steroids and growth factors, has the special interest.&#x0D; Aim of the work: to investigate the character of the expression of mRNAs of PR-A and PR-B isoforms of progesterone receptor in leiomyomas of different size and different intensity of cellular proliferation, and to elucidate the relationship between the PR-A and PR-B synthesis and activation of PI3K/Akt and MAPК signaling pathways in tumour tissue.&#x0D; Materials and Methods. The examination of the PR-A, PR-B, Akt, PTEN, ERK and Ki67 mRNAs expression in normal myometrium and leiomyoma tissues of 29 women with uterine leiomyoma was performed using RP-PCR.&#x0D; Results. It was found that in general the leiomyoma tissue was characterized by the high level of PR-A, PTEN and ERK mRNAs expression comparing to the miometrium. Distinctive characteristic of the small leiomyomas and leiomyomas with low proliferative activity was the highest synthesis of PR-A isoform of progesterone receptor. In large leiomyomas and in proliferating Ki67+ leiomymas the significant increase of ERK mRNA expression was seen.&#x0D; Conclusion. Activation of the PR-A isoform of progesterone receptor and MAPK/ERK signaling pathway associated with the inhibition of PI3K/Akt signaling due to high synthesis of PTEN plays the important role in mechanisms, regulating leiomyoma growth.

Berberine Inhibits Uterine Leiomyoma Cell Proliferation via Downregulation of Cyclooxygenase 2 and Pituitary Tumor-Transforming Gene 1.

We previously demonstrated that berberine (BBR) inhibits cell proliferation and induces apoptosis in a human uterine leiomyoma (UtLM) cell line but does not demonstrate a significant cytotoxic effect in a normal human uterine smooth muscle (UtSM) cell line. However, the mechanisms of this inhibition are unclear. Of note, cyclooxygenase 2 (COX2) and pituitary tumor-transforming gene 1 (PTTG1) are overexpressed in human uterine leiomyomata and are involved in the pathogenesis of uterine fibroids (UFs). We found that COX2 and PTTG1 were overexpressed in UtLM and that BBR decreased COX2 and PTTG1 expression in UtLM cells. Our data support that UtLM and UtSM are immortalized cell lines without phenotypic alterations from parental cell types and suggest that COX2 and PTTG1 are molecular targets for BBR. However, studies in these cell lines may not reveal all activities of BBR in vivo, and we therefore proceeded to test in this report the antitumor effects of BBR in an UF nude mouse xenograft model. When UF nude mice were killed at 7 weeks, tumor weight in controls was 45 ± 7 mg versus 20 ± 3 mg ( P < .05) in the low-dose (5 mg/kg) and 7 ± 3 mg ( P < .01) in the high-dose (10 mg/kg) BBR groups, respectively. Expression of proliferation markers, cell cycle-related genes, and UF-related genes was downregulated in tumors. No unusual behavioral changes and no signs of kidney or liver damage were observed in the animals with BBR treatment. In conclusion, our data suggests that (a) COX2 and PTTG1 are molecular targets for BBR and (b) BBR is potentially an effective and safe anti-UF agent.

Effects of Adlay Hull and Testa Ethanolic Extracts on the Growth of Uterine Leiomyoma Cells

Uterine leiomyoma has become a significant health concern affecting one in forth women in reproductive age in Taiwan, according to the Ministry of Health and Welfare. The associated symptoms can lead to reduced life quality and women ultimately seeking for surgical intervention. Adlay extract possesses anti-tumor activity and has shown to inhibit sarcoma cell proliferation and tumor progression. In present study, viability of ELT3 cells was tested to screen for different fractions of adlay extracts, as well as various phenolic compounds and flavonoids. The active adlay fractions and compounds were then tested on primary human leiomyoma cells. Cell cycle profiles of ELT3 cells were analyzed in order to determine the possible cause of growth-inhibition. Results showed that AHE-EA, ATE-Hex, and ATE-EA were able to inhibit the proliferation of ELT3 and primary leiomyoma cells. Among the tested compounds, Quercetin, nobiletin and eriodictyol, significantly decreased the viability of ELT3 and primary leiomyoma cells. Quercetin induced cell cycle arrest of ELT3 cells at Sub-G1 and G2/M phases. Adlay extracts exhibit growth-inhibitory effects on uterine leiomyoma cells. These effects may result from to the combined actions of different compounds, indicating a possibility of matrix effect from the compounds in adlay extract. Therefore, adlay extracts may be considered a feasible alternative therapeutic agent for leiomyoma.

Aspirin inhibits the proliferation of human uterine leiomyoma cells by downregulation of K‑Ras‑p110α interaction.

Aspirin has been confirmed as an effective antitumor drug in various cancers. However, the relationship between aspirin and uterine leiomyoma is still underexplored. Here, we explored the effects of aspirin on human uterine leiomyoma cells and provide insights into the underlying mechanisms. Cell Counting Kit-8 (CCK-8) and flow cytometry analysis showed that aspirin treatment inhibited cell proliferation and promoted cell cycle arrest at G0/G1 phase in a dose- and time‑dependent manner of human uterine leiomyoma cells. Further studies revealed that aspirin blocked the interaction between K-Ras and p110α by co-immunoprecipitation and immunofluorescence. Western blotting demonstrated K‑Ras‑p110α interaction was required for the effects of aspirin‑induced inhibition on cell growth and cell cycle transition via cell cycle regulators, including cyclinD1 and cyclin-dependent kinase 2 (CDK2). PI3K/Akt/caspase signaling pathway was involved in human uterine leiomyoma cell growth under aspirin treatment. Taken together, these results suggest that aspirin inhibited human uterine leiomyoma cell growth by regulating K‑Ras‑p110α interaction. Aspirin which targeting on interaction between K-Ras and p110α may serve as a new therapeutic drug for uterine leiomyoma treatment.

Silencing Med12 Gene Reduces Proliferation of Human Leiomyoma Cells Mediated via Wnt/β-Catenin Signaling Pathway.

Uterine fibroids, or leiomyoma, are the most common benign tumors in women of reproductive age. In this work, the effect of silencing the mediator complex subunit 12 (Med12) gene in human uterine fibroid cells was evaluated. The role of Med12 in the modulation of Wnt/β-catenin and cell proliferation-associated signaling was evaluated in human uterine fibroid cells. Med12 was silenced in the immortalized human uterine fibroid cell line (HuLM) using a lentivirus-based Med12 gene-specific RNA interference strategy. HuLM cells were infected with lentiviruses carrying Med12-specific short hairpin RNA (shRNA) sequences or a nonfunctional shRNA scrambled control with green fluorescence protein. Stable cells that expressed low levels of Med12 protein were characterized. Wnt/β-catenin signaling, sex steroid receptor signaling, cell cycle-associated, and fibrosis-associated proteins were measured. Med12 knockdown cells showed significantly (P < 0.05) reduced levels of Wnt4 and β-catenin proteins as well as cell proliferation, as compared with scrambled control cells. Med12 knockdown cells also showed reduced levels of cell cycle-associated cyclin D1, Cdk1, and Cdk2 proteins as well as reduced activation of p-extracellular signal-regulated kinase, p-protein kinase B, and transforming growth factor (TGF)-β signaling pathways as compared with scrambled control cells. Moreover, TGF-β-regulated fibrosis-related proteins such as fibronectin, collagen type 1, and plasminogen activator inhibitor-1 were significantly (P < 0.05) reduced in Med12 knockdown cells as compared with scrambled control cells. Together, these results suggest that Med12 plays a key role in the regulation of HuLM cell proliferation through the modulation of Wnt/β-catenin, cell cycle-associated, and fibrosis-associated protein expression.

Ulipristal acetate for uterine fibroid-related symptoms – a case report and review of the literature

Introduction: Uterine fibroids are the most commonly encountered benign uterine tumors in women of reproductive age. Although most women are asymptomatic (80%), fibroids, according to their type and location, can cause several symptoms and impact quality of life. To date, no medical treatment is able to eliminate fibroids. Ulipristal acetate (UPA) is an orally active synthetic selective progesterone receptor modulator (SPRM) characterized by a tissue-specific progesterone antagonist effect that reduces the proliferation of leiomyoma cells and induces apoptosis. Its pharmacological and pharmacodynamic characteristics, its efficacy and good tolerance make UPA a new important tool in the management of uterine fibroids. Aim: Analyze the efficacy of selective progesterone receptor modulators (SPRMs) in the treatment of uterine fibroid-related symptoms based in the description of a relevant clinical setting and review of the literature. Case report: A 32 years old woman, nulligesta, presented with excessive per vagina (PV) bleeding and symptomatic anemia. After hemodynamic stabilization and red blood cell transfusion, hysteroscopy was performed. The examination revealed multiple submucosal fibroids type 0, I (the larger ones with 33, 31 and 20 mm) and II (under 1 cm). She began treatment with ulipristal acetate with significant reduction of uterine abnormal bleeding after 1 month and amenorrhea was achieved after 3 months. At this time it was performed resectoscope. The hemoglobin prior to treatment was 13.6 g/dL. After medical and surgical treatment, she iniated oral contraception with good control of uterine bleeding after 1 year of initial therapy intervention. Discussion: No currently available medical treatment is able to eliminate fibroids. Therefore, there is no indication for medical treatment in the absence of symptoms. The choice of therapy is influenced by several factors, including the severity of symptoms (pain, bleeding), infertility related to fibroids, the tumor characteristics (volume, localization), the patient's age, wish to preserve the uterus, and wish to preserve fertility/desire to become pregnant. UPA can be used to control symptoms associated with fibroids and can allow surgery to be scheduled under better circumstances (e.g., a higher hemoglobin level, or myoma size reduction). Conclusion: Currently, no validated medical treatment is able to eliminate fibroids, and hence, surgery still represents the most effective treatment for symptomatic fibroids. UPA may be a good option for women seeking pregnancy, for women who wish to avoid surgery, or before surgery to reduce the invasiveness of the operation. New algorithms should be discussed, as there is no doubt that there is a place for medical therapy with SPRMs in the current fibroid's management.

MiR-139-5p regulates proliferation, apoptosis, and cell cycle of uterine leiomyoma cells by targeting TPD52.

BackgroundUterine leiomyoma is one of the most common benign tumors in women. It dramatically decreases the quality of life in the affected women. However, there is a lack of effective treatment paradigms. Micro-RNAs are small noncoding RNA molecules that are extensively expressed in organisms, and they are interrelated with the occurrence and development of the tumor. miR-139-5p was found to be downregulated in various cancers, but its function and mechanism in uterine leiomyoma remain unknown. The aim of this study was to investigate the role of miR-139-5p and its target gene in uterine leiomyoma.MethodsBy using a bioinformatic assay, it was found that TPD52 was a potential target gene of miR-139-5p. Then, expressions of miR-139-5p and TPD52 in uterine leiomyoma and adjacent myometrium tissues were evaluated by quantitative real-time polymerase chain reaction and Western blot. Proliferation, apoptosis, and cell cycle of uterine leiomyoma cells transfected by miR-139-5p mimics or TPD52 siRNA were determined.ResultsIt was observed that the expression of miR-139-5p in uterine leiomyoma tissues was significantly lower (P<0.001) than that in the adjacent myometrium tissues. Overexpression of miR-139-5p inhibited the growth of uterine leiomyoma cells and induced apoptosis and G1 phase arrest. Dual-luciferase reporter assay and Western blot validated that TPD52 is the target gene of miR-139-5p. Furthermore, downregulation of TPD52 by siRNA in uterine leiomyoma cells inhibited cell proliferation and induced cell apoptosis and G1 phase arrest.ConclusionData suggested that miR-139-5p inhibited the proliferation of uterine leiomyoma cells and induced cell apoptosis and G1 phase arrest by targeting TPD52.

Epigenetic regulation of transcription factor promoter regions by low-dose genistein through mitogen-activated protein kinase and mitogen-and-stress activated kinase 1 nongenomic signaling

BackgroundThe phytoestrogen, genistein at low doses nongenomically activates mitogen-activated protein kinase p44/42 (MAPKp44/42) via estrogen receptor alpha (ERα) leading to proliferation of human uterine leiomyoma cells. In this study, we evaluated if MAPKp44/42 could activate downstream effectors such as mitogen- and stress-activated protein kinase 1 (MSK1), which could then epigenetically modify histone H3 by phosphorylation following a low dose (1 μg/ml) of genistein.ResultsUsing hormone-responsive immortalized human uterine leiomyoma (ht-UtLM) cells, we found that genistein activated MAPKp44/42 and MSK1, and also increased phosphorylation of histone H3 at serine10 (H3S10ph) in ht-UtLM cells. Colocalization of phosphorylated MSK1 and H3S10ph was evident by confocal microscopy in ht-UtLM cells (r = 0.8533). Phosphorylation of both MSK1and H3S10ph was abrogated by PD98059 (PD), a MEK1 kinase inhibitor, thereby supporting genistein’s activation of MSK1 and Histone H3 was downstream of MAPKp44/42. In proliferative (estrogenic) phase human uterine fibroid tissues, phosphorylated MSK1 and H3S10ph showed increased immunoexpression compared to normal myometrial tissues, similar to results observed in in vitro studies following low-dose genistein administration. Real-time RT-PCR arrays showed induction of growth-related transcription factor genes, EGR1, Elk1, ID1, and MYB (cMyb) with confirmation by western blot, downstream of MAPK in response to low-dose genistein in ht-UtLM cells. Additionally, genistein induced associations of promoter regions of the above transcription factors with H3S10ph as evidenced by Chromatin Immunoprecipitation (ChIP) assays, which were inhibited by PD. Therefore, genistein epigenetically modified histone H3 by phosphorylation of serine 10, which was regulated by MSK1 and MAPK activation.ConclusionHistone H3 phosphorylation possibly represents a mechanism whereby increased transcriptional activation occurs following low-dose genistein exposure.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-016-0141-2) contains supplementary material, which is available to authorized users.

Locostatin, a disrupter of Raf kinase inhibitor protein, inhibits extracellular matrix production, proliferation, and migration in human uterine leiomyoma and myometrial cells.

To investigate the presence of Raf kinase inhibitor protein (RKIP) in human myometrium and leiomyoma as well as to determine the effect of locostatin (RKIP inhibitor) on extracellular matrix (ECM) production, proliferation, and migration in human myometrial and leiomyoma cells. Laboratory study. Human myometrium and leiomyoma. Thirty premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. Myometrial and leiomyoma tissues were used to investigate the localization and the expression level of RKIP through immunohistochemistry and Western blotting. Myometrial and leiomyoma cells were treated with locostatin (10μM) to measure ECM expression by real-time polymerase chain reaction, GSK3β expression by Western blotting, cell migration by wound-healing assay, and cell proliferation by MTT assay and immunocytochemistry. The expression of RKIP in human myometrial and leiomyoma tissue; ECM components and GSK3β expression, migration, and proliferation in myometrial and leiomyoma cells. RKIP is expressed in human myometrial and leiomyoma tissue. Locostatin treatment resulted in the activation of the mitogen-activated protein kinase (MAPK) signal pathway (ERK phosphorylation), providing a powerful validation of our targeting protocol. Further, RKIP inhibition by locostatin reduces ECM components. Moreover, the inhibition of RKIP by locostatin impaired cell proliferation and migration in both leiomyoma and myometrial cells. Finally, locostatin treatment reduced GSK3β expression. Therefore, even if the activation of MAPK pathway should increase proliferation and migration, the destabilization of GSK3β leads to the reduction of proliferation and migration of myometrial and leiomyoma cells. Our results indicate that RKIP may be involved in leiomyoma pathophysiology.

A selective serotonin 5-HT1B receptor inhibition suppresses cells proliferation and induces apoptosis in human uterine leiomyoma cells

ObjectiveUterine leiomyomas (fibroids or myomas) are the most common pelvic tumor in women with about 70% lifetime incidence rate. Currently, the only definitive treatment is surgery, causing undesirable side effects and negative impact on women’s quality of life, reproductive ability, and a substantial impact on healthcare costs. Therefore, curative medical treatments are needed to be developed. In this study, we investigated the impact of serotonin receptor 5-HT1B on cell proliferation and survival in human uterine leiomyoma cells (huLM). Study designThe impact of 5-HT1B receptor on cell proliferation, survival and apoptosis was investigated using a selective 5-HTR1B antagonist SB216641 in huLM cells, utilizing MTS, colony formation assay and Annexin V staining, respectively. Mechanisms of inhibition of cell proliferation, survival and induction of apoptosis were investigated by Western blot analysis after treatment with various doses of HT1B antagonist. Results5-HT1B receptor inhibition leads to a significant decrease in proliferation and colony formation in huLM cells, reduction of cyclin D1 and alpha-smooth muscle actin (α-SMA) expressions and the activity of Mitogen Activated Protein Kinase (MAPK) ERK and Elongation Factor 2 kinase (EF2K) pathways. 5-HT1B receptor blockage also induces apoptotic cell death by inducing cleavage of caspase-8, -9, and -3 and PARP. ConclusionOur findings show for the first time that 5-HT1B receptor promotes uterine leiomyoma cell survival and proliferation and its inhibition may be a potential therapeutic approach for human uterine leiomyomas. Thus, 5-HT1B expression and antagonists should be further investigated in leiomyoma tumors.

Targeted Adenoviral Vector Demonstrates Enhanced Efficacy for In Vivo Gene Therapy of Uterine Leiomyoma.

Gene therapy is a potentially effective non-surgical approach for the treatment of uterine leiomyoma. We demonstrated that targeted adenovirus vector, Ad-SSTR-RGD-TK/GCV, was highly effective in selectively inducing apoptosis and inhibiting proliferation of human leiomyoma cells in vitro while sparing normal myometrial cells. An in-vivo study, to compare efficacy and safety of modified adenovirus vector Ad-SSTR-RGD-TK/GCV versus untargeted vector for treatment of leiomyoma. Female nude mice were implanted with rat leiomyoma cells subcutaneously. Then mice were randomized into three groups. Group 1 received Ad-LacZ (marker gene), Group 2 received untargeted Ad-TK, and Group 3 received the targeted Ad-SSTR-RGD-TK. Tumors were measured weekly for 4 weeks. Then mice were sacrificed and tissue samples were collected. Evaluation of markers of apoptosis, proliferation, extracellular matrix, and angiogenesis was performed using Western Blot & Immunohistochemistry. Statistical analysis was done using ANOVA. Dissemination of adenovirus was assessed by PCR. In comparison with the untargeted vector, the targeted adenoviral vector significantly shrank leiomyoma size (P < 0.05), reduced expression of proliferation marker (PCNA) (P < 0.05), induced expression of apoptotic protein, c-PARP-1, (P < 0.05) and inhibited expression of extracellular matrix-related genes (TGF beta 3) and angiogenesis-related genes (VEGF & IGF-1) (P < 0.01). There were no detectable adenovirus in tested tissues other than leiomyoma lesions with both targeted and untargeted adenovirus. Targeted adenovirus, effectively reduces tumor size in leiomyoma without dissemination to other organs. Further evaluation of this localized targeted strategy for gene therapy is needed in appropriate preclinical humanoid animal models in preparation for a future pilot human trial.

Resveratrol inhibits proliferation of myometrial and leiomyoma cells and decreases extracellular matrix-associated protein expression

Uterine fibroids (leiomyomas) are the most common benign tumours in women during their reproductive age. Hyperplasia of uterine smooth muscle cells and abnormal deposition of extracellular matrix (ECM) are responsible for the development of uterine fibroids. Studies have shown that food rich in phytochemicals can prevent or treat leiomyoma. Therefore, the purpose of this study is to demonstrate the inhibitory effects of resveratrol on uterine fibroid cell growth and ECM-associated protein expression. We found that resveratrol inhibited the proliferation of leiomyoma cell line (ELT3) and uterine smooth muscle cell line (UtSMC) and affected the cell morphology of both cell lines, induced cell cycle arrest at S and G2/M phase, regulated cell apoptosis associated protein expression and induced cell apoptosis, and affected ECM-associated mRNA and protein expression. Our results suggest that resveratrol is potentially effective in preventing the hyperplasia of leiomyoma and uterine smooth muscle cells.

Raf Kinase Inhibitor Protein (RKIP) expression and function in human myometrium and leiomyoma

Many growth factors been identified in human myometrium and leiomyoma and activate multiple signaling pathways in order to regulate major cellular processes, including proliferation and fibrosis which are linked to uterine leiomyoma development and growth. The Raf kinase inhibitor protein (RKIP) has emerging roles as regulator of multiple signaling networks including mitogen activated protein (MAP) kinase cascade, as well as interaction with glycogen synthase kinase 3 (GSK3). In our study, we aimed to investigate the presence of RKIP in human myometrium and leiomyoma as well as to determine the effect of locostatin (RKIP inhibitor) on extracellular matrix (ECM) production, proliferation and migration in human myometrial and leiomyoma cells. Myometrial and leiomyoma tissues were used to investigate the localization and the expression level of RKIP through immunohistochemistry and western blotting. Myometrial and leiomyoma cells were treated with locostatin to measure ECM expression by real time PCR, GSK3b expression by western blotting, cell migration by wound-healing assay and cell proliferation by MTT assay. We found that RKIP is expressed in human myometrial and leiomyoma tissue. Locostatin treatment resulted in the activation of the MAPK signal pathway (ERK phosphorylation), providing a powerful validation of our targeting protocol. Further, RKIP inhibition by locostatin reduces ECM components. Moreover, the inhibition of RKIP by locostatin impaired cell proliferation and migration in both leiomyoma and myometrial cells. Finally, locostatin treatment reduced GSK3β expression. Therefore, even if the activation of MAPK pathway should increase proliferation and migration, the destabilization of GSK3β leads to the reduction of proliferation and migration of myometrial and leiomyoma cells.