The Potential of Transforming Growth Factor-beta Inhibitor and Vascular Endothelial Growth Factor Inhibitor as Therapeutic Agents for Uterine Leiomyoma.

Background: Uterine leiomyoma is the most common benign tumor in women of reproductive age, and it can cause infertility. The growth of uterine leiomyoma is mediated by various steroids and growth factors. The purpose of this study was to evaluate the expression of various growth factors in uterine leiomyoma. Additionally, comparing the effects of existing medication and specific growth factor inhibitors on leiomyoma and the normal myometrium, we aimed to see the potential of transforming growth factor-beta (TGF-ß) inhibitors and vascular endothelial growth factor (VEGF) inhibitors as therapeutic drugs for uterine leiomyoma. Methods: This in vitro study included uterine leiomyoma samples from 12 patients who underwent hysterectomy by laparoscopy or laparotomy at Seoul St. Mary's Hospital between May 2016 and March 2018. Normal myometrium and uterine leiomyoma tissue were obtained from each patient and the expression of growth factors was compared using immunohistochemical staining. After the primary culture of normal myometrial and leiomyoma cells, cell viability was evaluated following treatment with 100 nM ulipristal acetate (UPA) and mifepristone for 48 h. Western blot analysis was performed to determine the protein expression of each growth factor. Cell viability was determined following treatment with a 10-µM TGF-ß inhibitor (LY364947) and a 5-µM VEGF inhibitor (axitinib) for 24 h in cultured normal myometrium and leiomyoma cells. Results: Immunohistochemical staining revealed the significantly higher intensity of TGF-ß and VEGF in the leiomyoma tissue than in the normal myometrium (P < 0.05). Mifepristone treatment decreased VEGF expression by 62% in the leiomyoma cells (P < 0.05). According to the cell counting kit-8 (CCK-8) assay, cell viability was decreased after UPA, mifepristone, TGF-ß1 inhibitor, and VEGF inhibitor treatments in the normal myometrium and leiomyoma tissue. The effects of the TGF-ß1 inhibitor significantly differed between normal myometrium and leiomyoma tissue, with a greater decrease in cell survival in the leiomyoma tissue (P < 0.05). Post-hoc analysis showed that the TGF-ß1 and VEGF inhibitors had a greater inhibitory effect on leiomyoma tissue compared with that of UPA. Conclusion: TGF-ß and VEGF inhibitors significantly decreased the viability of uterine leiomyoma cells, showing stronger effects than the conventional drug, UPA. TGF-ß1 inhibitors affect both leiomyoma tissue and the normal uterus; thus, targeted local treatment rather than systemic treatment should be considered.

Effect of mistletoe on endometrial stromal cell survival and vascular endothelial growth factor expression in patients with endometriosis.

Research Question: To evaluate the effect of mistletoe on the cell viability of patients with endometriosis, the expression levels of vascular endothelial growth factor (VEGF) were measured, and the change in the expression level of VEGF following mistletoe treatment was recorded. Design: Forty reproductive-aged women with endometriosis (stage I/II [group 1, n=20], and stage III/IV [group 2, n=20]) were prospectively enrolled. Twenty women who underwent gynaecologic operations for benign conditions were selected as the control group. Both eutopic and ectopic endometrial tissues were obtained from the endometriosis patients. The endometrial tissues were cultured and the stromal cells were separated. The cells were cultured for 24 hours with peritoneal fluid from patients and controls with and without mistletoe supplementation (200 ng/mL), respectively. The MTT assay was used to assess cell viability, and VEGF expression was analysed by Western blotting and ELISA. Results: Using peritoneal fluid from endometriosis patients treated with mistletoe, we found that both eutopic and ectopic endometrial stromal cell viability increased after treatment with peritoneal fluid from patients with early-stage (I and II) endometriosis. After mistletoe treatment, the cell viability was decreased, in both eutopic and ectopic endometrial stromal cells in all stages of endometriosis. These findings were verified consistently by evaluating the expression and concentration of VEGF, a marker of angiogenesis. Conclusions: The present study showed that mistletoe can reduce the cell viability of endometrial stromal cells and the peritoneal fluid-induced elevation of VEGF in eutopic and ectopic endometrial stromal cells obtained from endometriosis patients, especially in the early stage. Mistletoe might have anti-angiogenic activity on endometrial stromal cells and thus is a potential candidate for the treatment of endometriosis.

Analysis of MED12 Mutation in Multiple Uterine Leiomyomas in South Korean patients.

Uterine leiomyomas are one of the most common benign gynecologic tumors, but the exact causes are not completely understood. In 2011, through DNA sequencing, MED12 mutation was discovered in approximately 71% of uterine leiomyomas. Several recent studies confirmed the high frequency of MED12 mutation in uterine leiomyoma. Nevertheless, no study has been done on MED12 mutation in the case of patients with multiple leiomyomas in a patient. The purpose of this study was to investigate the frequency of MED12 mutations in uterine leiomyomas of South Korean patients. In addition, we examined MED12 mutation in multiple leiomyomas in the same patients. Uterine leiomyoma tissues were obtained from symptomatic women who underwent hysterectomy or myomectomy for medically indicated reasons. We collected 60 uterine leiomyomas from 41 women. Tumor size ranged from 1 to 12cm. Patients' ages ranged from 25 to 55 years with an average of 38.4 years. Of the 60 tumors, 40 (66.67%) displayed MED12 mutation. Among the 41 patients, 14 patients had multiple leiomyomas and we analyzed those multiple leiomyomas. Three of them had the same mutations. Five of them, each leiomyoma had a different mutation. Two of them did not have mutation. Four of them had both mutation-positive and mutation-negative leiomyomas. In conclusion, we confirmed the high frequency of the MED12 mutation in uterine leiomyomas of South Korean patients. We also identified various MED12 mutation status in patients with multiple leiomyomas. This suggests that in a given patient, different tumors may have arisen from different cell origins and therefore it is supposed that occurrence of multiple leiomyoma in a single patient may not be caused by intrauterine metastasis or dissemination.

Effect of helixor A on natural killer cell activity in endometriosis.

BACKGROUND AND AIM: NK cells are one of the major immune cells in endometriosis pathogenesis. While previous clinical studies have shown that helixor A to be an effective treatment for endometriosis, little is known about its mechanism of action, or its relationship with immune cells. The aim of this study is to investigate the effects of helixor A on Natural killer cell (NK cell) cytotoxicity in endometriosis MATERIALS AND METHODS: We performed an experimental study. Samples of peritoneal fluid were obtained from January 2011 to December 2011 from 50 women with endometriosis and 50 women with other benign ovarian cysts (control). Peritoneal fluid of normal control group and endometriosis group was collected during laparoscopy. Baseline cytotoxicity levels of NK cells were measured with the peritoneal fluid of control group and endometriosis group. Next, cytotoxicity of NK cells was evaluated before and after treatment with helixor A. NK-cell activity was determined based upon the expression of CD107a, as an activation marker. RESULTS: NK cells cytotoxicity was 79.38±2.13% in control cells, 75.55±2.89% in the control peritoneal fluid, 69.59±4.96% in endometriosis stage I/II endometriosis, and 63.88±5.75% in stage III/IV endometriosis. A significant difference in cytotoxicity was observed between the control cells and stage III/IV endometriosis, consistent with a significant decrease in the cytotoxicity of NK cells in advanced stages of endometriosis; these levels increased significantly after treatment with helixor A; 78.30% vs. 86.40% (p=0.003) in stage I/II endometriosis, and 73.67% vs. 84.54% (p=0.024) in stage III/IV. The percentage of cells expressing CD107a was increased significantly in each group after helixor A treatment; 0.59% vs. 1.10% (p=0.002) in stage I/II endometriosis, and 0.79% vs. 1.40% (p=0.014) in stage III/IV. CONCLUSIONS: Helixor A directly influenced NK-cell cytotoxicity through direct induction of CD107a expression. Our results open new role of helixor A as an imune modulation therapy, or in combination with hormonal agents, for the treatment of endometriosis.

Comparison of the inhibitory effect of gonadotropin releasing hormone (GnRH) agonist, selective estrogen receptor modulator (SERM), antiprogesterone on myoma cell proliferation in vitro.

Uterine myomas are the most common gynecologic tumor in women of reproductive age. Treatment options of uterine myomas consist of surgical, medical and interventional therapy such as uterine artery embolization or myolysis. Given that it is the most common type of tumor in women of reproductive age, the treatment of uterine myomas must prioritize uterine conservation. There are several drugs for medical treatment of uterine myoma such as gonadotropin releasing hormone (GnRH) agonist, selective estrogen receptor modulator (SERM) and antiprogesterone. The objective of this study was to compare the effect of GnRH agonist, SERM, and antiprogesterone in the treatment of uterine myomas in vitro. The effect of drugs was evaluated through the cell viability assay in cultured leiomyoma cells, western blot analysis of proliferating cell nuclear antigen (PCNA), and BCL-2 protein expression. As a result, mifepristone single-treated group represents the most significant reduction in myoma cell viability and proliferation. When pretreated with leuprolide acetate, raloxifene shows more significant reduction in myoma cell viability and proliferation than mifepristone. This study suggests one of the possible mechanisms how medications act on uterine myoma, especially at the molecular level.

Antioxidant effect of estrogen on bovine aortic endothelial cells.

OBJECTIVE: This study discussed the role of estrogen as an antioxidant in the damage of vascular endothelial cells. DESIGN: We treated bovine aortic endothelial cells (bAEC) either with 1mM of H(2)O(2) alone or with 1 microM of 17beta-estradiol (E(2)) for 24h followed by 1mM of H(2)O(2) for 3h. The cell survival was evaluated by MTT assay, cellular apoptosis by fluorescence activated cell sorter (FACS) and Hoechst 33342 staining, oxidative stress by intracellular reactive oxygen species (ROS) and apoptosis after oxidative stress by western blotting for phospho-p38, p38, and Bcl-2. RESULTS: MTT assay showed that bAEC viability was reduced to 55.7+/-3.0% and 39.1+/-3.7% after 30 and 60 min of H(2)O(2) treatment, respectively. E(2) and H(2)O(2) treated cells did not show significant decrease in the cell survival. Similarly the FACS analysis and Hoechst 33342 stain showed that the latter decreased cellular apoptosis induced by H(2)O(2). Intracellular ROS increased by 181.6+/-68.9% in the former and by 37.0+/-3.9% in the latter (P<0.05). The expression of phospho-p38 mitogen-activated protein kinase (MAPK) was higher in the latter. CONCLUSIONS: E(2) mediates antioxidant effects on the oxidative stress induced by H(2)O(2). This antioxidant effect on bAEC may elucidate the scientific basis of hormone therapy for maintaining cardiovascular integrity in postmenopausal women.

Adherens junction-dependent PI3K/Akt activation induces resistance to genotoxin-induced cell death in differentiated intestinal epithelial cells.

The crypt-villi axis of intestinal mucosa maintains homeostasis by renewal of epithelia, and also exhibits different properties from undifferentiated to terminally differentiated cells. We investigated differential susceptibility to genotoxin-induced cell death, based on the degree of differentiation of epithelial cells, and its mechanism. Differentiation was induced by post-confluence culture in Caco-2 cells. Methyl methanesulfonate (MMS), a direct-acting DNA alkylating agent, was used for genotoxin-induced cell death. Compared to subconfluent Caco-2 cells, 7 days post-confluent cells showed resistance to MMS-induced cell death. With increasing expression of adherens junction components of E-cadherin and beta-catenin, E-cadherin and p-Akt expression increased in 7 days post-confluent Caco-2 cells, and in human intestinal tissue, expression of E-cadherin and p-Akt also increased in the upper portion of villi, compared to the crypt. Inhibition of cell-cell adhesion using EGTA decreased Akt phosphorylation, which was reversed by calcium restoration. Akt phosphorylation by calcium-mediated cell-cell adhesion was more prominent in differentiated cells. In addition, treatment of a PI3K inhibitor, LY294002, inhibited Akt phosphorylation by calcium-mediated cell-cell adhesion. Finally, the differential sensitivity to MMS-induced cell death between subconfluent and 7 days post-confluent Caco-2 cells was eliminated by inhibiting cell-cell adhesion or PI3K. Our data demonstrated that cell adhesion-mediated PI3K/Akt activation could be one of the important mechanisms of resistance to genotoxin-induced cell death in differentiated epithelial cells.

Antioxidant effects of equol on bovine aortic endothelial cells.

This study is to examine the effects of equol on the H(2)O(2)-induced death of bovine aortic endothelial cells (bAECs) and the mechanism of its protective effects. MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assay showed that in the control group, cell survival rate decreased significantly, each in proportion to the duration of the H(2)O(2) stimulation (P<0.05), but, in the equol-pretreated group, such decrease was not statistically significant. After Hoechst 33342 staining, in the equol-pretreated group the number of cells with apoptotic morphology decreased significantly. Equol pretreatment effectively inhibited the H(2)O(2)-induced cell death by the reduction of intracellular ROS production (P<0.05). Incubation of bAECs with equol increased the expression of phospho-p38 MAPK and Bcl-2 after the H(2)O(2) exposure compared with their expression without the equol pretreatment. Furthermore, SB203580 inhibited phospho-p38 MAPK expression and increased apoptotic cell death. This study proves equol has a significant antioxidant effect on the bAECs that were exposed to H(2)O(2).

Cellular differentiation-induced attenuation of LPS response in HT-29 cells is related to the down-regulation of TLR4 expression.

Intestinal epithelial cells not only present a physical barrier to bacteria but also participate actively in immune and inflammatory responses. The migration of epithelial cells from the crypt base to the surface is accompanied by a cellular differentiation that leads to important morphological and functional changes. It has been reported that the differentiation of colonic epithelial cells is associated with reduced interleukin (IL)-8 responses to IL-1beta. Although toll-like receptor 4 (TLR4) has been previously identified to be an important component of mucosal immunity to lipopolysaccharide (LPS) in the colon, little is known about the regulation of TLR4 in colonic epithelial cells during cellular differentiation. We investigated the effects of differentiation on LPS-induced IL-8 secretion and on the expression of TLR4. Differentiation was induced in colon cancer cell line HT-29 cells by butyrate treatment or by post-confluence culture and assessed by measuring alkaline phosphatase (AP) activity. IL-8 secretion was measured by ELISA, and TLR4 protein and mRNA expressions were followed by Western blot and RT-PCR, respectively. HT-29 cells were found to be dose-dependently responsive to LPS. AP activity increased in HT-29 cells by differentiation induced by treatment with butyrate or post-confluence culture. We found that IL-8 secretion induced by LPS was strongly attenuated in differentiated cells versus undifferentiated cells, and that cellular differentiation also attenuated TLR4 mRNA and protein expressions. Pretreating HT-29 cells with tumor necrosis factor (TNF)-alpha or interferon (INF)-gamma augmented LPS-induced IL-8 secretion and TLR4 expression. These TNF-alpha- or INF-gamma-induced augmentations of LPS response and TLR4 expression were all down-regulated by differentiation. Collectively, we conclude that cellular differentiation attenuates IL-8 secretion induced by LPS in HT-29 cells, and this attenuation is related with the down-regulation of TLR4 expression.