Human Endometrial Carcinoma Cell Line Research Articles

The interplay between the epithelial permeability barrier, cell migration and mitochondrial metabolism of growth factors and their inhibitors in a human endometrial carcinoma cell line

ABSTRACT It is known that there are abnormalities of tight junction functions, cell migration and mitochondrial metabolism in human endometriosis and endometrial carcinoma. In this study, we investigated the effects of growth factors and their inhibitors on the epithelial permeability barrier, cell migration and mitochondrial metabolism in 2D and 2.5D cultures of human endometrioid endometrial carcinoma Sawano cells. We also investigated the changes of bicellular and tricellular tight junction molecules and ciliogenesis induced by these inhibitors. The growth factors TGF-β and EGF affected the epithelial permeability barrier, cell migration and expression of bicellular and tricellular tight junction molecules in 2D and 2.5D cultures of Sawano cells. EW-7197 (a TGF-β receptor inhibitor), AG1478 (an EGFR inhibitor) and SP600125 (a JNK inhibitor) affected the epithelial permeability barrier, cell migration and mitochondrial metabolism and prevented the changes induced by TGF-β and EGF in 2D and 2.5D cultures. EW-7197 and AG1478 induced ciliogenesis in 2.5D cultures. In conclusion, TGF-β and EGF promoted the malignancy of endometrial cancer via interplay among the epithelial permeability barrier, cell migration and mitochondrial metabolism. EW-7197 and AG1478 may be useful as novel therapeutic treatments options for endometrial cancer.

Mechanism of E2F1 in the proliferation, migration, and invasion of endometrial carcinoma cells via the regulation of BMI1 transcription.

Endometrial carcinoma (EC) is the most prevalent gynecological cancer. Transcription factor (TF) regulates a large number of downstream target genes and is a key determinant of all physiological activities, including cell proliferation, differentiation, apoptosis, and cell cycle. The transcription factor E2F1 shows prominent roles in EC. BMI1 is a member of Polycomb suppressor Complex 1 (PRC1) and has been shown to be associated with EC invasiveness. It is currently unclear whether E2F1 can participate in the proliferation, migration, and invasion processes of EC cells by regulating BMI1 transcription. We investigated whether E2F1 could participate in the proliferation, migration, and invasion processes of EC cells by regulating BMI1 transcription, in order to further clarify the pathogenesis and etiology of EC, and provide reference for identifying potential therapeutic targets and developing effective prevention and treatment strategies for this disease. Human endometrial epithelial cells (hEECs) and human EC cell lines were selected. E2F1 expression was assessed by Western blot. E2F1 was silenced in AN3CA or overexpressed in HEC-1 by transfections, or E2F1 was silenced and BMI1 was overexpressed in AN3CA by cotransfection. Cell proliferation, migration, and invasion were detected by MTT, wound healing, and Transwell assays. The binding sites between E2F1 and BMI1 promoters were predicted through JASPAR website, and the targeted binding was verified by dual-luciferase report and ChIP assays. E2F1 was up-regulated in human EC cell lines, with its expression highest in AN3CA, and lowest in HEC-1. AN3CA invasion, migration, and proliferation were repressed by E2F1 knockdown, while those of HEC-1 cells were promoted by E2F1 overexpression. E2F1 overexpression increased the activity of wild type BMI1 reporter vector promoter, while this promotion was weakened after mutation of the predicted binding site in the BMI1 promoter. In the precipitated E2F1, BMI1 promoter site level was higher than that of IgG immunoprecipitant. BMI1 silencing suppressed AN3CA cell growth. BMI1 overexpression partially abrogated E2F1 silencing-inhibited EC cell growth. E2F1 promoted EC cell proliferation, invasion, and migration by promoting the transcription of BMI1.

Role of Ezrin/Radixin/Moesin in the Cell Surface Localization of Immune Checkpoint Molecule PD-L1 in Human Endometrial Carcinoma Cell Line.

Role of Ezrin/Radixin/Moesin in the Cell Surface Localization of Immune Checkpoint Molecule PD-L1 in Human Endometrial Carcinoma Cell Line.

Rhotekin 2 promotes progression of endometrial carcinoma by activating Akt/GSK3β signaling pathway


 
 
 
 Purpose: To investigate the expression of rhotekin 2 (RTKN2) in human endometrial carcinoma tissues and its role in cancer progression.
 Methods: Quantitative polymerase chain reaction (qPCR) and immunoblot assays were performed to determine the expression of RTKN2 in endometrial carcinoma tissues and cells. The effects of RTKN2 on cell proliferation were assessed using cell counting kit-8 and EdU assays, while its effects on cell cycle and apoptosis in endometrial carcinoma cells were evaluated by flow cytometry. The involvement of RTKN2 in activating Akt/glycogen synthase kinase-3 beta (GSK3β) pathway was characterized using an immunoblot assay. Tumor growth assays were performed to assess the effects of RTKN2 on the progression of endometrial carcinoma in vivo, while protein expression in tumor tissues was determined by an immunohistochemical assay.
 Results: High expression of RTKN2 was found in human endometrial carcinoma tissues and cell lines (p < 0.05). RTKN2 promoted the proliferation and cell cycle of endometrial carcinoma cells in vitro and suppressed apoptosis (p < 0.05). RTKN2 also activated Akt/GSK3β pathway in endometrial carcinoma cells and thus promoted tumor growth in vivo.
 Conclusion: The involvement of RTKN2 in the progression of endometrial carcinoma has been confirmed in this study. RTKN2 is thus a promising therapeutic target for patients with this disease.
 
 
 

Highly active PdPt bimetallic nanoparticles synthesized by one-step bioreduction method: Characterizations, anticancer, antibacterial activities and evaluation of their catalytic effect for hydrogen generation

Metallic nanoparticles (MNPs) have important applications in medicine and technology. Bimetallic NPs, which are among the metallic nanoparticles, are of great interest due to their properties. An innovative method by green synthesis has been developed to obtain bimetallic NPs. Aromatic plants are used in this synthesis method. One of the plants used for green synthesis is Nigella sativa and it has a unique place among plants for use as medicine. In this study, the synthesis of Palladium–Platinum bimetallic nanoparticles (PdPt NPs) and the catalytic, antibacterial, and anticancer activity of synthesized PdPt NPs by green synthesis method using Nigella sativa seed extract are reported. The synthesized PdPt NPs were characterized by Fourier Transform Infrared Spectrophotometer (FTIR), Transmission Electron Microscopy (TEM), X-ray diffraction (XRD), and UV–Vis spectrometry techniques. The catalytic activity of PdPt NPs was determined by performing sodium borohydride (NaBH4) hydrolysis experiments. According to the results obtained, Turnover Frequency (TOF), activation energy, entropy, and enthalpy values were found to be 1664.76 h−1, 13.93 kJ/mol, −119.02 J/mol.K, and 11.43 kJ/mol, respectively. It was determined that PdPt NPs are highly effective catalysts for hydrogen production. PdPt NPs (200 μg/mL) were determined to have antibacterial activity of 57.58%, 64.42%, 48.68%, and 58.77% against Escherichia coli, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, and Bacillus subtilis bacteria, respectively. In addition, the cytotoxic effects of PdPt NPs, MTT against human breast cancer cell line (MDA-MB-231), human endometrial carcinoma cell line (Ishikawa, ISH), human cervical cancer cell line (HeLa), L929-Murine fibroblast cell line test, and IC50 values were calculated. The IC50 values of PdPt NPs applied against MDA-MB-231, ISH, and HeLa cancer cell lines were calculated as 9.1744 ± 1.566 μg/mL, 12.2431 ± 1.132 μg/mL, 18.1963 ± 1.730 μg/mL, respectively. No significant cytotoxic effect was observed in healthy L929-murine fibroblasts. Green synthesis of PdPt NPs was determined to have significant advantages over chemical approaches. The biogenic PdPt NPs synthesized in this study suggest the design of bio-based bimetallic catalysts with high catalytic performance to prevent environmental pollution.

Curcumin Inhibits the Growth and Induces Apoptosis of Human Endometrial Cancer Cells

The objective of this study was to analyze the mechanism of curcumin inhibiting the growth and inducing apoptosis of human endometrial carcinoma cell line. Human endometrial carcinoma cell line was cultured in vitro and all cells were divided into control group, curcumin low, medium and high treatment group, the concentrations were 5 mg/l, 10 mg/l and 20 mg/l, respectively. Compared with the non-medicated control group, the proliferation of human endometrial carcinoma cell treated with curcumin at different concentrations for 24, 48 and 72 h was significantly inhibited and with the increase of drug concentration, the inhibition rate was significantly increased in a dose-dependent manner (p<0.05). After treatment of human endometrial carcinoma cell with curcumin 5 mg/l, 10 mg/l and 20 mg/l, the ratio of growth 0/ growth 1 was significantly lower than that of the control group, the ratio of growth 2/mitosis phase was significantly higher than that of the control group and the ratio of growth 2/mitosis phase was gradually increased with the increase of drug concentration, which was dose-dependent (p<0.05). After treatment of human endometrial carcinoma cells with curcumin 5 mg/l, 10 mg/l and 20 mg/l, the apoptosis rate gradually increased and with the increase of drug concentration, the apoptosis rate increased sequentially (p<0.05). After treatment of human endometrial carcinoma cell with curcumin 5 mg/l, 10 mg/l and 20 mg/l, compared with the control group, the protein level of apoptosis factor Livin was significantly decreased and the protein level of caspase-3 was significantly increased in each group (p<0.05). The levels of Livin protein and caspase-3 protein were dose-dependent with curcumin concentration. It was concluded that curcumin has obvious inhibitory effect on human endometrial cancer cells and can induce cell cycle arrest, promote apoptosis, which is concentration dependent. The related reason may be through inhibiting Livin protein expression and increasing caspase-3 protein level.

P–403 Sodium tungstate increases embryo adhesion through a direct effect on endometrial cells

Abstract Study question Does sodium tungstate treatment induce a change in endometrial cells’ capacity to implant trophoblasts? Summary answer Administration of sodium tungstate to endometrial cells increases trophoblast adhesion. What is known already Sodium tungstate (ST) has shown its capacity to modulate the activity of cytokines, such as leptin, an activator of an obligatory signalling cascade in the embryo-implantation process. STAT3, a signal transducer molecule critical for the embryo implantation process, is also known to be activated by ST. Still, ST’s effect on implantation using biological systems has never been studied. Embryo implantation process and endometrium roles are complicated to study in vivo due to a lack of animal models and appropriate techniques. In vitro techniques using immortalised cell lines allows a first approach to study early implantation stages, such as embryo adhesion. Study design, size, duration An in vitro study was carried out using a human endometrial carcinoma cell line (HEC–1-A) treated with sodium tungstate for 24 and 48h, and choriocarcinoma cell spheroids (JAr). Different times of treatment and concentrations were studied. Each experiment was performed in triplicate. Participants/materials, setting, methods Confluent endometrial HEC–1-A cultures were treated with ST at concentrations (0–150mM) and withaferin A (1mM), negative control for embryo adhesion. After the treatment period, HEC–1-A cultures were washed with ST-free culture medium to eliminate ST. Immediately, 15 JAr trophoblast spheroids were added to cultures and coincubated with gentle agitation for 30, 60 and 90 minutes. An inverted light microscope was used to count adhered and floating spheroids, and determine the trophoblast adherence ratio. Main results and the role of chance HEC–1-A cells treated with ST showed normal morphology and growth at all doses except 150mM. At the highest dose tested, the cells’ culture was still viable (negative blue trypan staining) and maintained morphology, but the adhesion to the plate surface was affected. Doses from 0.15 to 15mM were used to perform adhesion assays. HEC–1-A cells treated with ST for 24h showed an increased capacity to adhere JAr trophoblast spheroids. Adhesion rates reached significant differences at doses of 1.5 and 15mM after 60 and 90 minutes of coincubation. After 90 minutes, untreated cells reached 32.8% adhesion rate, while 1.5 and 15mM ST-treated cells reached 54.6% and 53.4% respectively (p &amp;lt; 0.05 ST vs untreated). Thus, the increment of trophoblast adhesion rate induced by ST reached 66%. Lower adhesion rates were observed after 60 minutes of coincubation but were also significant with a relative increase of 49.1% at 1.5mM and 50.5% at 1.5mM when compared with untreated cells (p &amp;lt; 0.05) Longer treatments (48h) showed similar trends to 24h-treatments, but with a lower extent of ST effect on HEC–1-A receptivity. Maximum adhesion rates were also observed at 90 minutes of coincubation and 1.5 and 15mM doses. The Mean adhesion rate increase was &amp;gt;40% with both doses. Limitations, reasons for caution: The current study is the first approach to evaluate sodium tungstate effect on endometrium using an in vitro model. Future research using in vivo models should be performed to assess sodium tungstate effect on endometrium receptivity and its potential as a fertility treatment. Wider implications of the findings: We conclude that the direct effect of sodium tungstate on endometrial cells increases embryo adhesion rate. These results open a new research line to a potential treatment in human reproduction management with sodium tungstate to solve the unmet need of inducing embryo implantation. Trial registration number Not applicable

The anti-tumoral effects of different local anesthetics on human endometrial carcinoma cell line: An in-vitro study

Background: There is accumulating evidence on the inhibitory effect of local anesthetic agents on metastasis and tumor recurrence on various cancer types. In this study, we aimed to test the effects of lidocaine, bupivicaine and prilocaine on cellular viability and apoptosis on human endometrial adenocarcinoma cell lines (Ishikawa cells). Material and methods: Human Endometrial adenocarcinoma cells (Ishikawa cells) were exposed to different concentrations of local anesthetic agents for 24, 48, and 72 hours. The cell viability assay was performed via MTT and concentrations corresponding to 50% effective concentration (EC50) of cells were determined for each agent. Apoptosis was evaluated via commercial Biocolor ApoPercentage assay. Results: All of the three local anesthetics reduced the amount of viable tumor cells in a dose-dependent manner. The EC50 values (mM) at 24, 48 and 72 h incubation periods were 11.67, 1.915, 2.114 for lidocaine 11.4, 5.21, 3.12 for bupivacaine 7.64 mM, 7.3 mM, and 2.83 mM, for prilocaine respectively. Conclusion: Lidocaine, bupivacaine, and prilocaine have anti-tumoral effects on Ishikawa cell lines. All of the tested agents induced apoptosis and necrosis at high concentrations in a dose dependent-manner.

Glyphosate induces epithelial mesenchymal transition-related changes in human endometrial Ishikawa cells via estrogen receptor pathway

Glyphosate based herbicides are the most commonly used herbicide in the world. We aimed to determine whether glyphosate (Gly) induces epithelial mesenchymal transition (EMT) - related changes in a human endometrial carcinoma cell line (Ishikawa cells), and whether the estrogen receptor (ER) pathway is involved in these changes. Ishikawa cells were exposed to Gly (0.2 μM and 2 μM) or 17β-estradiol (E2: 10−9 M). We detected that Gly increased cell migration and invasion ability compared to vehicle, as did E2. Moreover, a down regulation of E-cadherin mRNA expression was determined in response to Gly, similar to E2-effects. These results show that Gly promotes EMT-related changes in Ishikawa cells. When an ER antagonist (Fulvestrant: 10−7 M) was co-administrated with Gly, all changes were reversed, suggesting that Gly might promote EMT-related changes via ER-dependent pathway. Our results are interesting evidences of Gly effects on endometrial cancer progression via the ER-dependent pathway.

Localization of Tricellular Tight Junction Molecule LSR at Midbody and Centrosome During Cytokinesis in Human Epithelial Cells.

Epithelial integrity and barrier function are maintained during cytokinesis in vertebrate epithelial tissues. The changes in localization and the roles of tricellular tight junction molecule lipolysis-stimulated lipoprotein receptor (LSR) during cytokinesis are not well known, although new tricellular tight junctions form at the flank of the midbody during cytokinesis. In this study, we investigated the changes in localization and the role of LSR at the midbody and centrosome during cytokinesis using human endometrial carcinoma cell line Sawano, comparing the tricellular tight junction molecule tricellulin; bicellular tight junction molecules occludin, claudin-7, zonula occludens-1, and cingulin; and the epithelial polarized related molecules apoptosis-stimulating of p53 protein 2, PAR3, and yes-associated protein. During cytokinesis induced by treatment with taxol, the epithelial barrier was maintained and the tricellular tight junction molecules LSR and tricellulin were concentrated at the flank of the acetylated tubulin-positive midbody and in γ-tubulin-positive centrosomes with the dynein adaptor Hook2, whereas the other molecules were localized there as well. All the molecules disappeared by knockdown using small interfering RNAs. Furthermore, by the knockdown of Hook2, the epithelial barrier was maintained and most of the molecules disappeared from the centrosome. These findings suggest that LSR may play crucial roles not only in barrier function but also in cytokinesis.

Bu Shen Yang Xue Prescription Has Treating Effect on Endometrial Cancer through FSH/PI3K/AKT/Gankyrin/HIF-α/cyclinD 1 Pathway in Ishikawa Cells.

Background. The formulation of Bu Shen Yang Xue (BSYX) has been clinically used in treating gynecologic disease in China, especially for the development of the endometrium. Endometrial carcinoma is the most common malignant tumor of the female genital tract in developed countries. And few studies have been reported on the antitumor activity of BSYX. Therefore, this study aimed to investigate the effect of BSYX on endometrial cancer and make an initial discussion of the underlining mechanisms in Ishikawa cells. Methods and Results. Firstly, 60 SPF female nude mice were randomly divided into control group, model group, BSYX group, and positive group. The models of subcutaneous tumor xenograft of nude mice were established by injection of human endometrial carcinoma cell line Ishikawa tumor cell suspension. Compared with model group, BSYX reduced effectively tumor volume and changed pathological feature in mice tumor issue. Meanwhile, proteins from tumor issues were detected by western blot analysis. The protein levels of follicle-stimulating hormone receptor (FSHR), p-Akt/Akt, Gankyrin, and cyclinD1 in the model group were higher than those in control group but the expression in BSYX group was lower than that in the model group. The hypoxia inducible factor alpha (HIF-α) protein level in the model group was lower than those in control group and upregulated in BSYX group. In addition, Ishikawa cells were cultured and then exposed to follicle-stimulating hormone (FSH), LY294002, a highly selective PI3K inhibitor and serum containing BSYX, respectively. LY294002 and BSYX markedly decreased the cancer cell viability and migration ability and increased the apoptosis rate. FSH promoted the cancer cell ability and migration ability. LY294002 and BSYX evidently downregulated the proteins levels of FSHR, p-Akt/Akt, Gankyrin, and cyclinD1 and upregulated the expression of HIF-α protein, and FSH was on the opposite. Conclusions. Taken together, our results showed that the formulation of BSYX had antitumor effect on endometrial cancer in vivo and in vitro and was related with FSH/PI3K/AKT/Gankyrin/HIF-α/cyclinD1 transduction pathway.

Relaxin 2/RXFP1 Signaling Induces Cell Invasion via the β-Catenin Pathway in Endometrial Cancer.

Relaxin is known to play an important role in animal pregnancies, including those of humans. It is suggested that relaxin induces aggressive cell growth and invasiveness in several types of cancer, including endometrial cancer. However, the mechanisms of relaxin remain largely unclear. In this study, we examined the effects of relaxin 2 (RLN2), the major circulating relaxin in humans, on human endometrial carcinoma cell lines. RLN2 treatment induced invasion in HEC-1B and Ishikawa cells. RLN2-induced cell invasion was significantly decreased by transfection of relaxin receptor 1 (RXFP1) siRNAs. The β-catenin inhibitor, XAV939, also significantly inhibited the RLN2-induced cell invasions. Both a decrease of cadherin expression and an increase of β-catenin phosphorylation were observed in response to the RLN2 treatment in HEC-1B and Ishikawa cells. We then examined RLN2 and RXFP1 expression in 80 human endometrioid endometrial carcinoma tissues. RLN2 immunoreactivity was detected in the human endometrial carcinoma cells and had a correlative tendency with histological grade and RXFP1. These results suggest that adherens junctions in cancer cells are weakened by the breakdown of the cadherin/catenin complex, which is induced by β-catenin phosphorylation via RLN2/RXFP1 signaling.

Effects of growth hormone on pregnancy rates of patients with thin endometrium.

To investigate whether growth hormone (GH) could improve pregnancy rates of patients with thin endometrium by clinical study and laboratory experiments. Ninety-three patients were randomized to either the GH-received group (40) or the routine exogenous administration of estrogens control group (53) for clinical study. The human endometrial carcinoma cell line RL95-2 was used for testing the role of GH with Western blot and real-time PCR by exposure to various concentrations of GH (0.1 nM,1 nM,10 nM,100 nM). Patients treated with GH had a significantly (P<0.05) greater endometrium thickness on day 3 (7.87±0.72 vs 6.34±0.86), higher implantation rates (24.4% vs 10.5%) and greater clinical pregnancy rates (42.5% vs 18.9%) compared with the control group. No adverse events were associated with the use of GH. Administration of GH significantly up-regulated the expression of VEGF, ItgB3 and IGF-I expression in RL95-2 cells at both mRNA and protein levels (P<0.05). AG490, an inhibitor of JAK2, nearly completely inhibited the up-regulative effect of GH through the JAK2-STAT5 pathway, and GH-induced effects could be mediated through autocrine IGF-I together with its hepatic counterpart. IGF-I mRNA was detected in the RL95-2 cells. GH may improve pregnancy outcomes of patients with thin endometrium who undergo frozen embryo transfer by acting on human endometrial cells to promote proliferation and vascularization and to up-regulate receptivity-related molecular expression.

Long noncoding RNA HOTAIR mediates the estrogen-induced metastasis of endometrial cancer cells via the miR-646/NPM1 axis.

LncRNA homeobox (HOX) transcript antisense intergenic RNA (HOTAIR) has been confirmed to be involved in the tumorigenic progression of endometrial carcinoma (EC). However, the molecular mechanisms of HOTAIR in EC are not fully elucidated. The expression of HOTAIR and miR-646 in human EC tissues was determined by qRT-PCR. The effect of miR-646 on EC cells was assessed by the cell viability, migration, and invasion using CCK-8 assays and transwell assays. RNA-binding protein immunoprecipitation assays and RNA pull-down assays were performed to explore the interaction between HOTAIR and miR-646. The regulation of miR-646 on nucleophosmin 1 (NPM1) was tested using luciferase reporter assays. MiR-646 expression was significantly decreased both in human EC tissues ( n = 23) and cell lines (Ishikawa and HEC-1-A) compared with the control. Moreover, miR-646 expression was negatively related to HOTAIR in human EC tissues ( n = 23). Our results also showed that miR-646 overexpression considerably attenuated the E2-promoted viability, migration, and invasion of Ishikawa and HEC-1-A cells in vitro. In addition, HOTAIR was confirmed to regulate the viability, migration, and invasion of EC cells through negative regulating miR-646. More importantly, we also demonstrated that NPM1 was the target of miR-646, and HOTAIR promoted NPM1 expression through interacting with miR-646 in EC cells. Taken together, our findings presented that HOTAIR could regulate NPM1 via interacting with miR-646, thereby governing the viability, migration, and invasion of EC cells.

Development of an In Vitro Model to Screen CYP1B1-Targeted Anticancer Prodrugs

Cytochrome P450 1B1 (CYP1B1) is an anticancer therapeutic target due to its overexpression in a number of steroid hormone–related cancers. One anticancer drug discovery strategy is to develop prodrugs specifically activated by CYP1B1 in malignant tissues to cytotoxic metabolites. Here, we aimed to develop an in vitro screening model for CYP1B1-targeted anticancer prodrugs using the KLE human endometrial carcinoma cell line. KLE cells demonstrated superior stability of CYP1B1 expression relative to transiently transfected cells and did not express any appreciable amount of cognate CYP1A1 or CYP1A2, which would have compromised the specificity of the screening assay. The effect of two CYP1B1-targeted probe prodrugs on KLE cells was evaluated in the absence and presence of a CYP1B1 inhibitor to chemically “knock out” CYP1B1 activity (CYP1B1 inhibited). Both probe prodrugs were more toxic to KLE cells than to CYP1B1-inhibited KLE cells and significantly induced G0/G1 arrest and decreased the S phase in KLE cells. They also exhibited pro-apoptotic effects in KLE cells, which were attenuated in CYP1B1-inhibited KLE cells. In summary, a KLE cell–based model has been characterized to be suitable for identifying CYP1B1-targeted anticancer prodrugs and should be further developed and employed for screening chemical libraries.

Caudatin targets TNFAIP1/NF-κB and cytochrome c/caspase signaling to suppress tumor progression in human uterine cancer.

Caudatin, a C-21 steroidal glyco-side isolated from Chinese herbs, has a long history of use for the treatment of multiple diseases, including cancers. However, the precise mechanisms of actions of caudatin in human uterine cancer cells remain unclear. In this study, we investigated the molecular mechanisms by which caudatin inhibits cell growth in human cervical carcinoma cell line (HeLa) and endometrial carcinoma cell line (HEC-1A). Treatment with caudatin promoted cell morphology change, inhibited cell proliferation, colony formation, migration and spheroid formation, and induced cell apoptosis. Our results showed that the expression of tumor necrosis factor; α-induced protein 1 (TNFAIP1) was downregulated in uterine cancer cells and tissues compared to paired adjacent non-tumor uterine tissues. Further molecular mechanism study showed that caudatin can directly regulate TNFAIP1 expression in a concentration-dependent manner and also associated with the downregulation of NF-κB and upregulation of BAX/BcL-2 ratio and caspase-3. Moreover, we found that overexpression of TNFAIP1 inhibits the growth and invasion, and induces apoptosis in uterine cancer cells through inhibition of the NF-κB pathway, suggesting that TNFAIP1 may act as a potential therapeutic target for the treatment of cancer. We found that caudatin inhibited tumorigenicity and upregulated TNFAIP1 in vivo. Taken together, caudatin impacts on cell proliferation, migration and apoptosis of uterine cancer cells by regulating several carcinogenesis-related processes, including a novel mechanism involving the targeting of TNFAIP1/NF-κB signaling. Our findings provide new insights into understanding the anticancer mechanisms of caudatin in human uterine cancer therapy.

Mifepristone inhibites the migration of endometrial cancer cells through regulating H19 methylation

To investigate the effect and mechanism of mifepristone on the migration of human endometrial carcinoma cells. A human endometrial carcinoma cell line, Ishikawa cells, was cultured in vitro and treated with mifepristone at different concentrations. Wound healing assay was applied to detect the migration of Ishikawa cells. RT-PCR and methylation-specific PCR (MSP) were used to detect the levels of H19 mRNA and its DNA methylation. Western-blot was used to detect the expressions of HMGA2 and epithelial to mesenchymal transition (EMT) related proteins. When treated with different concentrations of mifepristone for 48 hours, the width of scratch of the the control group, the 5 mg/L and the 10 mg/L mifepristone treatment groups were (4.18±0.07)mm, (4.68±0.07)mm, and(4.99±0.07)mm, respectively (P<0.05 for all) and treated with mifepristone for 72 hours, those were(3.46±0.07)mm, (4.29±0.07)mm, and(4.78±0.04)mm, respectively (P<0.05 for all). In the Ishikawa cells, mifepristone suppressed the transcriptional level of H19 through enhancing its promoter methylation, which resulted in inhibited expressions of HMGA2 and vimentin and increased expression of E-cadherin in a time- and concentration-dependent manner. Mifepristone inhibits the migration of endometrial carcinoma cells partially through methylation-induced of transcriptional inhibition of H19, which results in the down-regulation of HMGA2 and vimentin and upregulation of E-cadherin.

Targeting cancer stem cells with p53 modulators.

Cancer stem cells (CSC) typically over-express aldehyde dehydrogenase (ALDH). Thus, ALDHbright tumor cells represent targets for developing novel cancer prevention/treatment interventions. Loss of p53 function is a common genetic event during cancer development wherein small molecular weight compounds (SMWC) that restore p53 function and reverse tumor growth have been identified. Here, we focused on two widely studied p53 SMWC, CP-31398 and PRIMA-1, to target ALDHbright CSC in human breast, endometrial and pancreas carcinoma cell lines expressing mutant or wild type (WT) p53. CP-31398 and PRIMA-1 significantly reduced CSC content and sphere formation by these cell lines in vitro. In addition, these agents were more effective in vitro against CSC compared to cisplatin and gemcitabine, two often-used chemotherapeutic agents. We also tested a combinatorial treatment in methylcholantrene (MCA)-treated mice consisting of p53 SMWC and p53-based vaccines. Yet using survival end-point analysis, no increased efficacy in the presence of either p53 SMWC alone or with vaccine compared to vaccine alone was observed. These results may be due, in part, to the presence of immune cells, such as activated lymphocytes expressing WT p53 at levels comparable to some tumor cells, wherein further increase of p53 expression by p53 SMWC may alter survival of these immune cells and negatively impact an effective immune response. Continuous exposure of mice to MCA may have also interfered with the action of these p53 SMWC, including potential direct interaction with MCA. Nonetheless, the effect of p53 SMWC on CSC and cancer treatment remains of great interest.

Effects of silencing MTA1 gene by RNA interference on invasion and metastasis of endometrial carcinoma.

The study aimed at the silence effect of MAT1 gene and proteins in Ishikawa cell of human endometrial carcinoma cell line through RNAi mediated by siRNA and also discussed the effect of MTA1 gene silence on the invasion and metastasis of endometrial carcinoma. According to the principle of designing siRNA sequence, siRNA aiming at MTA1 gene (MTA1-siRNA) and siRNA used for negative control (Control-siRNA) were designed and synthesized and Ishikawa cell was transfected by transfection reagents. RT-PCR method as well as western blot was used, respectively, to detect the MTA1 mRNA and protein expression of stably transfected cells. Transwell method and scarification experiment were adopted to detect the invasion and metastasis of Ishikawa cells. The expression results of MTA1 on the levels of mRNA and protein showed that the expression level in transfected MTA1-siRNA group was obviously lower than that in non-transfected group and transfected control-siRNA group (p <0.05), while there was no significant difference between non-transfected group and transfected control-siRNA group (p > 0.05). Effective interference on the expression of MTA1 gene remarkably lowered the invasion and metastasis of endometrial carcinoma Ishikawa cells. RNAi aiming at MTA1 can effectively inhibit the expression of MTA1 in endometrial carcinoma Ishikawa cells and the effective silence of MTA1 can weaken the invasion and metastasis of Ishikawa cells, which provides a new strategy for gene therapy of endometrial carcinoma and an experimental basis for inhibiting the invasion and metastasis of endometrial carcinoma.

TGF-βI Regulates Cell Migration through Pluripotent Transcription Factor OCT4 in Endometriosis.

Transforming growth factor (TGF-β)/TGF-β receptor signal is known to promote cell migration. Up-regulation of TGF-β in serum/peritoneal fluid and increased levels of pluripotent transcription factor OCT4 in endometriotic tissues are frequently observed in patients with endometriosis. However, the mechanisms underlying how TGF-β/TGF-β receptor and OCT4 affect endometriotic cell migration still remain largely unknown. Therefore, endometriotic tissue with high cell migratory capacity were collected from patients with adenomyotic myometrium (n = 23) and chocolate cyst (n = 24); and endometrial tissue with low cell migratory capacity in normal endometrium or hyperplastic endometrium (n = 8) were collected as the controls. We found the mRNA levels of TGF-β receptor I (TGF-β RI) and OCT4 were significantly higher in the high-migratory ectopic endometriotic tissues than those of the low-migratory normal or hyperplastic endometrium. Positive correlations between TGF-β RI and OCT4, and either TGF-β RI or OCT4 with migration-related genes (SNAIL, SLUG and TWIST) regarding the mRNA levels were observed in human endometriotic tissues. TGF-βI dose-dependently increased the gene and protein levels of OCT4, SNAIL and N-Cadherin (N-CAD) and silencing of endogenous OCT4 significantly suppressed the TGF-βI-induced expressions of N-CAD and SNAIL in primary human endometriotic stromal cells and human endometrial carcinoma cell lines RL95-2 and HEC1A. Furthermore, TGF-βI significantly increased the migration ability of endometriotic cells and silencing of OCT4 dramatically suppressed the TGF-βI-induced cell migration activity evidenced by wound-closure assay, transwell assay, and confocal image of F-actin cellular distribution. In conclusion, the present findings demonstrate that the niche TGF-β plays a critical role in initiating expressions of pluripotent transcription factor OCT4 which may contribute to the ectopic endometrial growth by stimulating endometrial cell migration. These findings would be useful for developing therapeutic strategies targeting TGF-β-OCT4 signaling to prevent endometriosis in the future.