HEC-1A Cells Research Articles

LncRNA ROR promotes proliferation of endometrial cancer cells via regulating Notch1 pathway.

The aim of this study was to investigate the effects of long non-coding ribonucleic acid regulator of reprogramming (lncRNA ROR) on the proliferation and apoptosis of endometrial cancer (EC) cells, and to explore its possible underlying mechanism. The expression levels of lncRNA ROR and Notch1 in EC tissues were detected via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The changes in Notch1 protein were detected via Western blotting. Subsequently, the regulatory mechanism of lncRNA ROR on Notch1 was analyzed using Luciferase reporter gene assay. Moreover, the changes in cell proliferation and apoptosis were determined through cell counting kit-8 (CCK-8) assay and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, respectively. Both lncRNA ROR and Notch1 were highly expressed in EC tissues (p<0.05). After overexpression of lncRNA ROR, HEC-1A cells had significantly enhanced proliferation (p<0.05) and weakened apoptosis (p<0.05). Meanwhile, the mRNA and protein levels of Notch1 rose remarkably compared with those in control group (p<0.05). Luciferase reporter gene assay revealed that lncRNA ROR could bind to the Notch1 regulatory factor miR-34a and inhibit its activity. LncRNA ROR regulates the proliferation and apoptosis of EC cells via promoting the expression of Notch1 protein.

SPOCK2 Affects the Biological Behavior of Endometrial Cancer Cells by Regulation of MT1-MMP and MMP2

Abnormal expression of SPARC (osteonectin), cwcv and kazal-like domains proteoglycan 2 (SPOCK2) plays a significant role in the development and progression of various human cancers, yet a relationship between SPOCK2 and endometrial cancer (EC) has not been reported. Here, we assessed the potential role and mechanism by which SPOCK2 acts in the pathogenesis and progression of EC. First, protein expression of SPOCK2 in EC tissue from patients was detected by immunohistochemistry and associated clinical data were analyzed. Then, HEC-1A and Ishikawa cells were transfected with an adenoviral vector containing an SPOCK2 recombinant fragment and the biological behavior of transfected cells was observed. Finally, the expression of membrane type 1 matrix metalloproteinase (MT1-MMP) and MMP2 in the transfected cells was detected by Western blot and zymography gel assay to analyze the effect of SPOCK2 on the regulation of the MT1-MMP/MMP2 pathway. We found that there was significantly less SPOCK2 protein expression in the EC tissue than in the normal endometrium tissue, and lack of SPOCK2 protein expression in EC tissue was associated with distant metastasis and myometrial invasion. Upregulation of SPOCK2 in HEC-1A and Ishikawa cells inhibited cell proliferation, invasion, adhesion, and apoptosis. Upregulation of SPOCK2 inhibited the expression of MT1-MMP and MMP2 and activation of MMP2 in HEC-1A and Ishikawa cells. Collectively, our data indicated that SPOCK2 contributed to the progression of EC by regulating the biological behavior of cancer cells, which is achieved partly through regulating protein expression of MT1-MMP and MMP2 and activation of MMP2.

Effects of Vitamin D Analogs Alfacalcidol and Calcitriol on Cell Proliferation and Migration of HEC1A Endometrial Adenocarcinoma Cells

Endometrial carcinoma is the most diagnosed among infiltrating tumor of the female genital tract. Vitamin D has antiproliferative and immunomodulatory properties besides its classical effect on calcium and phosphate. We aimed to demonstrate whether alfacalcidol and calcitriol have any effect on proliferation and migration. Endometrial adenocarcinoma HEC1A was used as a cancer cell line. The effect of alfacalcidol (1α-hydroxyvitamin D3) and calcitriol (1α,25-dihydroxyvitamin D3) on proliferation was demonstrated by real-time cell analysis device and migration was shown by a wound healing model. 10−11–10−9M alfacalcidol and calcitriol reduced both proliferation and migration. In contrast, the high concentration of alfacalcidol and calcitriol (10−8–10−6M) increased proliferation and migration. The proliferative effects of alfacalcidol (0–12 h) immediately started earlier than calcitriol (12–48 h). In contrast, the antiproliferative effects of calcitriol (12–24 h) have begun earlier than alfacalcidol (48–60 h). While the high concentrations of alfacalcidol and calcitriol increased the migration relatively earlier (12–24 h), low concentrations decreased the migration at late times (24–48 h). Lower concentrations of vitamin D prevent proliferation and migration in endometrial adenocarcinoma HEC1A cells. In contrast, high concentrations of vitamin D increase proliferation and migration.

Mixed-ligand complexes of lanthanides derived from an α-hydroxycarboxylic acid (benzilic acid) and 1,10-phenanthroline: Physicochemical properties and anticancer activity

The reactions of [Ln(NO3)3·6H2O] (Ln = Pr and Sm), [SmCl3·6H2O] and [Nd(OAc)3·xH2O] with benzilic acid (H2ben) and 1,10-phenanthroline (phen) as an auxiliary ligand in basic media yielded isostructural, nine-coordinate complexes (monoclinic system, space group P21/n) of the type [Ln(Hben)3(phen)2(H2O)]·DMF·H2O (where Ln = Pr(III), Nd(III) and Sm(III)). In the structures, the monodeprotonated Hben− ligand connects the metal centres monodentately through the deprotonated carboxylate oxygen, and bidentately via the hydroxy oxygen and the carboxylate group oxygen. Additional stability of the crystal structures is provided by intra- and inter-molecular OH⋯O interactions, CH⋯O contacts, CH⋯π and parallel-displaced aromatic π⋯π interactions. The electronic absorption spectra of the complexes show π → π* transitions of the bidentate aromatic phen, as well as Laporte-forbidden 4f-4f transitions. Anticancer screen indicated that phen, and the Pr(III) and Nd(III) complexes, significantly reduced MCF-7 cell proliferation (p < 0.05). 1,10-Phenanthroline also exerted cytotoxic effects in HEC-1A cells (p < 0.05).

Lanthanide complexes with N-(2,6-dimethylphenyl)oxamate: Synthesis, characterisation and cytotoxicity

The complexation reactions of Ce(III), Nd(III), Gd(III) and Er(III) ions with the chelating/ bridging monoanionic ligand N-(2,6-dimethylphenyl)oxamate (Hpma−) in basic media were performed in view of the potential applications of oxamate derivatives as cytotoxic agents. The coordination compounds were characterised by different physico-chemical techniques: elemental analysis, conductivity measurements, IR, 1H NMR and UV–Vis-NIR spectroscopy, and X-ray crystallography. The anionic Hpma− ligand was obtained through conversion of the proligand ethyl (2,6-dimethylphenylcarbamoyl)formate (Hdmp). The reactions afforded lanthanide(III)–oxamate coordination polymers of the formulae: {[Ln(Hpma)3(MeOH)(H2O)]∙2MeOH}n (Ln = Ce (1) and Nd (2)), {[Gd2(Hpma)6(MeOH)4]∙6MeOH}n (3), {[Er2(Hpma)6(MeOH)(H2O)3]∙2MeOH}n (4) and [Ln2Na2(Hpma)8(EtOH)(H2O)6]n (Ln = Nd (5) and Gd (6)). The polymeric complexes feature Ln-Hpma moieties bridged by μ2-η1:η1:η1 Hpma− ligands, giving one-dimensional zig-zag chains of the LnOCOLn type. Atomic charge analysis and the MEP map of the Hpma− moiety using the DFT/B3LYP method were consistent with the chelating and bridging modes of the anionic ligand through all the oxygen atoms. An evaluation of the cytotoxic activities of the metal salts, the proligand and the novel lanthanide complexes on MCF-7, HEC-1A and THP-1 cells revealed that only the rare-earth metal salts [Ce(NO3)3·6H2O] and [Nd(NO3)3·6H2O] showed moderate cytotoxicity against MCF-7 and HEC-1A cells, respectively.

Synthesis, characterisation and anticancer activity screening of lanthanide(III) acetate complexes with benzohydrazone and nicotinohydrazone ligands

Centrosymmetric, acetato-bridged dinuclear coordination compounds with the formulae [La2(Hphen)2(OAc)4(H2O)2]·DMF·H2O (1), [Ln2(HNic)2(OAc)4(H2O)2]·DMF·H2O (Ln = La (2) and Nd (3)) and [Ln2(HNic)2(OAc)4(H2O)2]·DMF (Ln = Er (4) and Yb (5)) (H2phen = (E)-N'-(2-hydroxybenzylidene)benzohydrazide and H2Nic = (E)-N'-(2-hydroxybenzylidene)nicotinohydrazide) were isolated from the reactions of lanthanide acetates and hydrazones, and characterised by elemental analyses, IR spectroscopy, UV–Vis spectroscopy, X-ray diffraction studies and SHAPE 2.1. The nine-coordinate distorted spherical capped square antiprism complexes 1–3 crystallise in the triclinic space group P-1, while the eight-coordinate triangular dodecahedron (D2d) Er(III) and Yb(III) complexes belong to the monoclinic system (space group P21/c). Geometry optimisation of the monoanionic forms of the hydrazone ligands (Hphen– and HNic–) were performed at the DFT/B3LYP/aug-cc-pVTZ level of theory. Natural population analysis (NPA) and molecular electrostatic potential (MEP) maps indicated that the most preferred sites for electrophilic attack in the anionic ligands were the phenolate and carbonyl oxygen atoms, and the azomethine nitrogen atoms. Cytotoxic studies of the compounds on breast cancer (MCF-7), endometrial carcinoma (HEC-1A) and human monocytic (THP-1) cell lines using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay revealed that the hydrazone ligands and complexes 1–4 are partially cytotoxic against MCF-7 cells, while the Schiff bases and complexes 3–5 significantly inhibit cell growth in HEC-1A cells.

LEFTY2/endometrialbleeding-associated factor up-regulates Na+Coupled Glucose Transporter SGLT1 expression and Glycogen Accumulation in Endometrial Cancer Cells.

LEFTY2 (endometrial bleeding associated factor; EBAF or LEFTYA), a cytokine released shortly before menstrual bleeding, is a negative regulator of cell proliferation and tumour growth. LEFTY2 down-regulates Na+/H+ exchanger activity with subsequent inhibition of glycolytic flux and lactate production in endometrial cancer cells. Glucose can be utilized not only for glycolysis but also for glycogen formation. Both glycolysis and glycogen formation require cellular glucose uptake which could be accomplished by the Na+ coupled glucose transporter-1 (SGLT1; SLC5A1). The present study therefore explored whether LEFTY2 modifies endometrial SGLT1 expression and activity as well as glycogen formation. Ishikawa and HEC1a cells were exposed to LEFTY2, SGLT1 and glycogen synthase (GYS1) transcript levels determined by qRT-PCR. SGLT1, GYS1 and phospho-GYS1 protein abundance was quantified by western blotting, cellular glucose uptake from 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) uptake, and cellular glycogen content utilizing an enzymatic assay and subsequent colorimetry. As a result, a 48-hour treatment with LEFTY2 significantly increased SGLT1 and GYS1 transcript levels as well as SGLT1 and GYS1 protein abundance in both Ishikawa and HEC1a cells. 2-NBDG uptake and cellular glycogen content were upregulated significantly in Ishikawa (type 1) but not in type 2 endometrial HEC1a cells, although there was a tendency of increased 2-NBDG uptake. Further, none of the effects were seen in human benign endometrial cells (HESCs). Interestingly, in both Ishikawa and HEC1a cells, a co-treatment with TGF-β reduced SGLT1, GYS and phospho-GYS protein levels, and thus reduced glycogen levels and again HEC1a cells had no significant change. In conclusion, LEFTY2 up-regulates expression and activity of the Na+ coupled glucose transporter SGLT1 and glycogen synthase GYS1 in a cell line specific manner. We further show the treatment with LEFTY2 fosters cellular glucose uptake and glycogen formation and TGF-β can negate this effect in endometrial cancer cells.

MicroRNA-155 Suppresses the Translation of p38 and Impairs the Functioning of Dendritic Cells in Endometrial Cancer Mice.

BackgroundDendritic cells (DCs) are reported to play an important role in activating the anti-tumor immune responses. p38 MAPK14 signaling plays an important role in controlling their activity. Here, we identified that miR-155 suppressed the translation of p38 and impaired the functioning of dendritic cells in endometrial cancer.MethodsHEC1A endometrial cancer cell lines were used for the study which was transfected in the C57BL/6 mice. Murine bone marrow-derived dendritic cells (BMDCs) were isolated from the mice. Target prediction was done by TargetScan which was confirmed by RT-PCR analysis. The protein expression was carried by Western blot analysis. Levels of IL-12 were evaluated by ELISA. Mice injected with HEC1A cells were subjected to tumor challenge study.ResultsOn screening the binding sites of p38 MAPK14 gene, miR-155 was found to bind the 3ʹUTR directly and blocked its translation. The levels of miR-155 were upregulated in dendritic cells and RAW264.7 cells, miR-155 showed inhibitory effect on expression levels of p38. In dendritic cells, miR-155 was found to regulate the expression of IL-12, also miR-155 inhibitor stimulated the differentiation of Th1 cells in mice induced with endometrial cancer. In dendritic cells, miR-155 inhibited the expression of p38 gene and decreased their ability to interfere in tumor growth.ConclusionThe study concludes suppressive role of miR-155 in the process of dendritic cells mediated anti-tumor immunity, also inhibiting miR-155 provides a novel strategy for countering endometrial cancer.

In vitro anti-HIV activity of some Indian medicinal plant extracts

BackgroundHuman Immunodeficiency Virus (HIV) persists to be a significant public health issue worldwide. The current strategy for the treatment of HIV infection, Highly Active Antiretroviral Therapy (HAART), has reduced deaths from AIDS related disease, but it can be an expensive regime for the underdeveloped and developing countries where the supply of drugs is scarce and often not well tolerated, especially in persons undergoing long term treatment. The present therapy also has limitations of development of multidrug resistance, thus there is a need for the discovery of novel anti-HIV compounds from plants as a potential alternative in combating HIV disease.MethodsTen Indian medicinal plants were tested for entry and replication inhibition against laboratory adapted strains HIV-1IIIB, HIV-1Ada5 and primary isolates HIV-1UG070, HIV-1VB59 in TZM-bl cell lines and primary isolates HIV-1UG070, HIV-1VB59 in PM1 cell lines. The plant extracts were further evaluated for toxicity in HEC-1A epithelial cell lines by transwell epithelial model.ResultsThe methanolic extracts of Achyranthes aspera, Rosa centifolia and aqueous extract of Ficus benghalensis inhibited laboratory adapted HIV-1 strains (IC80 3.6–118 μg/ml) and primary isolates (IC80 4.8–156 μg/ml) in TZM-bl cells. Methanolic extract of Strychnos potatorum, aqueous extract of Ficus infectoria and hydroalcoholic extract of Annona squamosa inhibited laboratory adapted HIV-1 strains (IC80 4.24–125 μg/ml) and primary isolates (IC80 18–156 μg/ml) in TZM-bl cells. Methanolic extracts of Achyranthes aspera and Rosa centifolia, (IC801-9 μg/ml) further significantly inhibited HIV-1 primary isolates in PM1cells. Methanolic extracts of Tridax procumbens, Mallotus philippinensis, Annona reticulate, aqueous extract of Ficus benghalensis and hydroalcoholic extract of Albizzia lebbeck did not exhibit anti-HIV activity in all the tested strains. Methanolic extract of Rosa centifolia also demonstrated to be non-toxic to HEC-1A epithelial cells and maintained epithelial integrity (at 500 μg/ml) when tested in transwell dual-chamber.ConclusionThese active methanolic extracts of Achyranthes aspera and Rosa centifolia, could be further subjected to chemical analysis to investigate the active moiety responsible for the anti-HIV activity. Methanolic extract of Rosa centifolia was found to be well tolerated maintaining the epithelial integrity of HEC-1A cells in vitro and thus has potential for investigating it further as candidate microbicide.

Fatostatin inhibits the development of endometrial carcinoma in endometrial carcinoma cells and a xenograft model by targeting lipid metabolism

Endometrial carcinoma is a type of gynecological cancer that originates in the endometrial epithelial tissue. Due to its high proliferation and ability to invade muscle tissue, it is one of the most common malignant tumors in the female reproductive system. Fatostatin is a small molecule non-sterol diarylthiazole derivative that acts as a chemical inhibitor of the sterol regulatory-element binding protein (SREBP) pathway. Previous studies have shown that fatostatin has an anti-tumor effect in some cancers. In this study, we investigated the effect of fatostatin on the growth, proliferation, apoptosis, migration and cell cycle of human endometrial carcinoma cells (HEC-1A and AN3 CA cells) using cholecystokinin (CCK) −8 method, clonogenicity assay, wound closure assay, Transwell migration assay and flow cytometer. We also examined its effect on the expression of apoptosis-associated protein (Caspase-3, Caspase-8 and Caspase-9) and level of lipid metabolism-related proteins, free fatty acid, and total cholesterol in cells. The growth of endometrial carcinoma xenografts was measured to confirm the effect of fatostatin in vivo. Our results showed that fatostatin inhibited the growth and proliferation of human endometrial carcinoma cells, changed their cell cycle and induced apoptosis. Based on the preliminary animal experiments, fatostatin also exhibited antitumor activity. The present study adds a new dimension to our understanding of the antitumor effects of fatostatin and provides an experimental basis for its use, and supports its potential value for clinical application.

MicroRNA-191 regulates endometrial cancer cell growth via TET1-mediated epigenetic modulation of APC

Endometrial cancer (EC) is a common gynecological malignancy with relatively favourable prognosis, although alternative diagnostic and therapeutic options remain to be explored for advanced disease. Recent studies enabled to apply microRNAs (miRs) to clinical cancer management as promising diagnostic and therapeutic biomarkers. We here aimed to identify proliferation-associated miRNAs and characterize their functions in EC cells. Our small RNA-sequencing analysis showed that miR-191 is abundantly expressed in HEC-1A and Ishikawa EC cells along with the high expression of miR-182, which was previously characterized as an EC proliferation-related miRNA in EC. We showed that miR-191 was upregulated in EC tissues than in adjacent normal tissues and its knockdown repressed EC cell proliferation. In silico miRNA target screening identified that ten-eleven translocation 1 (TET1) is one of the putative miR-191 targets. TET1 expression could be downregulated by miR-191 through the mRNA-miRNA interaction in the 3'-untranslated region of TET1. In line with TET1 functions as a methylcytosine dioxygenase, which removes genome-wide DNA methylation marks, decreased TET1 expression resulted in hypermethylation in the promotor region of tumour suppressor adenomatous polyposis coli. Taken together, miR-191 could function as an oncogenic miRNA in EC and serve as a prospective diagnostic and therapeutic target for advanced disease.

Sirtuin 2 in Endometrial Cancer: A Potential Regulator for Cell Proliferation, Apoptosis and RAS/ERK Pathway.

Objective:The present study aimed to explore the function of sirtuin 2 (SIRT2) on cell proliferation, apoptosis, rat sarcoma virus (RAS)/ extracellular signal-regulated kinase (ERK) pathway in endometrial cancer (EC).Methods:SIRT2 expression in human EC cell lines and human endometrial (uterine) epithelial cell (HEEC) line was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot. SIRT2 knock-down and control knock-down plasmids were transfected into HEC1A cells, respectively; SIRT2 overexpression and control overexpression plasmids were transfected into Ishikawa cells, respectively. After transfection, SIRT2, HRas proto-oncogene, GTPase (HRAS) expressions were evaluated by RT-qPCR and western blot. ERK and phosphorylated ERK (pERK) expressions were evaluated by western blot. Meanwhile, cell proliferation and cell apoptosis were measured.Results:Compared to normal HEEC cell line, SIRT2 mRNA and protein expressions were increased in most human EC cell lines (including HEC1A, RL952 and AN3CA), while were similar in Ishikawa cell line. In HEC1A cells, SIRT2 knock-down decreased cell proliferation but increased apoptosis. In Ishikawa cells, SIRT2 overexpression induced cell proliferation but inhibited apoptosis. For RAS/ERK pathway, SIRT2 knock-down reduced HRAS and inactivated pERK in HEC1A cells, whereas SIRT2 overexpression increased HRAS and activated pERK in Ishikawa cells, suggesting that SIRT2 was implicated in the regulation of RAS/ERK pathway in EC cells.Conclusion:SIRT2 contributes to the EC tumorigenesis, which appears as a potential therapeutic target.

Cell Proteins Interacting with the Human Immunodeficiency Virus in Immunoblotting can be Detected by R5- or X4- Tropic Human Immunodeficiency Virus Particles.

Introduction:The present study reported a new immunoblot assay, with revelation by R5- or X4-whole free human immunodeficiency virus (HIV) particles or recombinant gp160.Materials and Methods:The assay was optimized to identify cell proteins interacting with HIV. Whole cell lysates were prepared from peripheral blood lymphocytes (PBLs), dendritic cells (DC), monocyte-derived macrophage (MDM), and Henrietta Lacks (Hela, wild-type or transfected with DC-specific intracellular adhesion molecule-3-Grabbing Non-Integrin, HeLa) and Human endometrial cells (HEC-1A) lines; HIV particles used were the R5-tropic HIV-1JRCSF and the X4-tropic HIV-1NDK.Results:Experiments with PBL lysates and both viruses demonstrated different bands, including a unique band at 105–117 kDa in addition to nonspecific bands. The 105–117 kDa band migrated at the same level of that observed in controls using total PBL lysate and anti-CD4 mAb for detection and thus likely corresponds to the cluster difference (CD) 4 complex. Blots using lysates of DCs, MDM, HeLa cell line, and HEC-1A cell line allowed identifying several bands that positions were similar to that seen by recombinant gp160 or whole R5- or X4-HIV particles.Conclusion:Blot of whole lysates of various HIV target cells is recognized by free HIV particles and allows identifying a wide range of HIV-interacting cell proteins. Such optimized assay could be useful to recognize new cellular HIV attachment proteins.

Relationship between polycomb-group protein BMI-1 and phosphatases regulating AKT phosphorylation level in endometrial cancer.

The PI3K/AKT pathway is frequently activated in endometrial carcinoma. BMI‐1 (B‐lymphoma Mo‐MLV insertion region 1) protein affects expression of PTEN (phosphatase and tensin homolog) in some cancers, but its significance for endometrial tumorigenesis is not known. The objective of this study was to determine the relationship between BMI‐1 and expression of factors affecting AKT (protein kinase B) phosphorylation level in endometrial cancer. The expression of proteins and mRNAs was investigated in endometrial cancer specimens and samples of non‐neoplastic endometrial tissue by Western blot and RT‐PCR, respectively. The impact of BMI‐1 down‐regulation on AKT phosphorylation and expression of genes coding for several phosphatases were studied in HEC1A cells. The results showed that BMI‐1 depletion caused increase in PHLPP1 and PHLPP2 (PH domain and leucine‐rich repeat protein phosphatases 1/2) expression and decrease in phospho‐AKT (pAKT) level. In more advanced tumours with higher metastatic potential, the expression of BMI‐1 was lower compared to tumours less advanced and without lymph node metastasis. There were significant inverse correlations between BMI‐1 and PHLPPs, especially PHLPP1 in normal endometrial samples. The inverse correlation between BMI‐1 and PHLPP1/PHLPP2 expression was observed in PTEN positive but not PTEN negative cancers. Low PHLPP2 expression in tumours predicted poorer overall survival. BMI‐1 impacts on AKT phosphorylation level in endometrial cells by regulation of PHLPP expression.

Extracellular Vesicles-Based Biomarkers Represent a Promising Liquid Biopsy in Endometrial Cancer.

Tumor-derived extracellular vesicles (EVs) are secreted in large amounts into biological fluids of cancer patients. The analysis of EVs cargoes has been associated with patient´s outcome and response to therapy. However, current technologies for EVs isolation are tedious and low cost-efficient for routine clinical implementation. To explore the clinical value of circulating EVs analysis we attempted a proof-of-concept in endometrial cancer (EC) with ExoGAG, an easy to use and highly efficient new technology to enrich EVs. Technical performance was first evaluated using EVs secreted by Hec1A cells. Then, the clinical value of this strategy was questioned by analyzing the levels of two well-known tissue biomarkers in EC, L1 cell adhesion molecule (L1CAM) and Annexin A2 (ANXA2), in EVs purified from plasma in a cohort of 41 EC patients and 20 healthy controls. The results demonstrated the specific content of ANXA2 in the purified EVs fraction, with an accurate sensitivity and specificity for EC diagnosis. Importantly, high ANXA2 levels in circulating EVs were associated with high risk of recurrence and non-endometrioid histology suggesting a potential value as a prognostic biomarker in EC. These results also confirmed ExoGAG technology as a robust technique for the clinical implementation of circulating EVs analyses.

Hypoxia and hyperglycaemia determine why some endometrial tumours fail to respond to metformin

BackgroundHigh expression of Ki67, a proliferation marker, is associated with reduced endometrial cancer-specific survival. Pre-surgical metformin reduces tumour Ki-67 expression in some women with endometrial cancer. Metformin’s anti-cancer activity may relate to effects on cellular energy metabolism. Since tumour hypoxia and glucose availability are major cellular redox determinants, we evaluated their role in endometrial cancer response to metformin.MethodsEndometrial cancer biopsies from women treated with pre-surgical metformin were tested for the hypoxia markers, HIF-1α and CA-9. Endometrial cancer cell lines were treated with metformin in variable glucose concentrations in normoxia or hypoxia and cell viability, mitochondrial biogenesis, function and energy metabolism were assessed.ResultsIn women treated with metformin (n = 28), Ki-67 response was lower in hypoxic tumours. Metformin showed minimal cytostatic effects towards Ishikawa and HEC1A cells in conventional medium (25 mM glucose). In low glucose (5.5 mM), a dose-dependent cytostatic effect was observed in normoxia but attenuated in hypoxia. Tumours treated with metformin showed increased mitochondrial mass (n = 25), while in cultured cells metformin decreased mitochondrial function. Metformin targets mitochondrial respiration, however, in hypoxic, high glucose conditions, there was a switch to glycolytic metabolism and decreased metformin response.ConclusionsUnderstanding the metabolic adaptations of endometrial tumours may identify patients likely to derive clinical benefit from metformin.

FXYD5/Dysadherin, a Biomarker of Endometrial Cancer Myometrial Invasion and Aggressiveness: Its Relationship With TGF-β1 and NF-κB Pathways.

Objective: Endometrial cancer (EC) is the second most common gynecological cancer worldwide. Myometrial invasion (MI) is a key event in EC dissemination. This study aimed to evaluate FXYD5/dysadherin (FXYD5/Dys) expression in EC tissue and uterine aspirate (UA) biopsies and to assess molecular/functional changes associated with its expression in cellular models.Methods: FXYD5/Dys messenger RNA (mRNA) levels were determined in EC tissue and UA biopsies. FXYD5/Dys expression was evaluated in EC RNAseq data from The Cancer Genome Atlas (TCGA) and GENEVESTIGATOR tools. FXYD5/Dys impact on E-cadherin expression and cell behavior was assessed in EC Hec1a cells treated with transforming growth factor (TGF)-β1, stably transfected with ETV5, and transiently transfected with FXYD5/Dys small interfering RNA (siRNA) or pcDNA3-FXYD5/Dys plasmid.Results: FXYD5/Dys was associated with EC aggressiveness, finding high mRNA levels in tumors depicting MI > 50%, Grade 3, and intermediate/high risk of recurrence. FXYD5/Dys was highly expressed at the tumor invasive front compared to the superficial area. Most results were recapitulated in UA biopsies. FXYD5/Dys modulation in Hec1a cells altered cell migration/adhesion and E-cadherin expression. TGF-β1 treatment of Hec1a cells induced FXYD5/Dys expression. TCGA-UCEC RNAseq analysis revealed a positive correlation between FXYD5/Dys, TGF-β1, and plasminogen activator inhibitor (PAI)-1 mRNA levels. FXYD5/Dys induced nuclear factor (NF)-κB pathway activation in Hec1a cells. FXYD5/Dys mRNA levels positively correlated with transcriptional activation of NF-κB p65-regulated genes. Survival analysis revealed patient segregation into low- and high-risk groups, the latter depicting the highest FXYD5/Dys, PAI-1, tumor necrosis factor (TNF)-α, and TGF-β1 mRNA levels and shorter survival rates.Conclusion: FXYD5/Dys is a novel biomarker of EC progression related to TGF-β1 and NF-κB pathways that collectively promote tumor dissemination and result in poor patient prognosis.

LncRNA NEAT1 promotes endometrial cancer cell proliferation, migration and invasion by regulating the miR-144-3p/EZH2 axis.

BackgroundEndometrial cancer (EC) is one of the most common gynaecological tumours in the worldwide. Long non-coding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) promotes cell proliferation, migration and invasion in EC cells. However, the molecular mechanisms of NEAT1 in EC have not been fully clarified. We conducted this study to reveal the function of NEAT1 in EC tissues and cell lines.Materials and methodsCancer and adjacent tissues were collected from EC patients. HEC-1A and Ishikawa cells were cultured in vitro. NEAT1 expression was downregulated by transfecting small hairpin RNA (shRNA) and miR-144-3p was overexpressed by transfecting miR-144-3p mimics. Cell proliferation was detected by MTT assay and colony formation assay. Cell migration and invasion abilities were assessed by transwell assay. A dual-luciferase reporter assay was used to verify the relationship among NEAT1, EZH2, and miR-144-3p. The expression level of EZH2 was measured by Western blot and qPCR.ResultsNEAT1 was highly expressed in EC tissues and cells. Knockdown of NEAT1 inhibited the proliferation, migration and invasion of EC cells. Additionally, NEAT1 acted as a ceRNA of miR-144-3p, leading to EZH2 upregulation. Overexpression of miR-144-3p suppressed the proliferation and invasion of EC cells.ConclusionsNEAT1 promotes EC cells proliferation and invasion by regulating the miR-144-3p/EZH2 axis.

Clinical and biological significance of EZH2 expression in endometrial cancer

ABSTRACTThe objective of this study was to examine the clinical significance of EZH2 expression and the therapeutic efficacy of its silencing in endometrial cancer. EZH2 expression in clinical samples was evaluated using a tissue microarray and correlated with clinical outcomes. The biological roles of EZH2 were assayed in vitro and in vivo. Gene expression was examined to reveal the molecular mechanism underlying the roles of EZH2 in endometrial cancer. We found that EZH2 overexpression was significantly correlated with disease-free and overall survival of patients with endometrial cancer. EZH2 silencing resulted in decreased cell viability and invasiveness, and increased apoptosis. In addition, EZH2 silencing enhanced the cytotoxicity of taxanes and cisplatin in Hec-1A and Ishikawa endometrial cancer cells. EZH2 silencing using small-interfering RNA (siRNA) incorporated into chitosan nanoparticles (siRNA/CN) induced a significant anti-tumor effect compared with that observed in controls (66.6% reduction in Hec-1A cells and 63.2% reduction in Ishikawa cells, p < .05 for both). Moreover, EZH2 siRNA/CN in combination with taxanes produced more robust anti-tumor effects versus those induced by monotherapies (77.0% for Hec-1A cells and 57.7% for Ishikawa cells, p < .05 for both). These results were associated with decreased angiogenesis and cell proliferation, and enhanced apoptosis. Genomic analyses revealed that EZH2 silencing decreased the expression levels of many genes associated with tumor growth, including PRDX6. Collectively, these results support EZH2 as an attractive target for the therapeutic management of endometrial cancer.

Antitumor Effects of MRS5698, a Cordycepin Derivative, on Endometrial Cancer Cells

Endometrial cancer drug treatments often produce undesirable effects. Thus, discovering new drugs with fewer side effects is required. Cordycepin is a constituent of Cordyceps sinensis, which has been proven to inhibit tumor growth by stimulating the adenosine A3 receptor (A3R). However, cordycepin is rapidly degraded by adenosine deaminase (ADA) and has a clinically unacceptable short half-life. One of its derivatives, MRS5698, was predicted to exhibit antitumor effects with a poor affinity to ADA by our previous validated in silico experiments. The purpose of this study was to explore the possibilities of using MRS5698 as a novel antitumor agent through experiments on Ishikawa and HEC-1A cells. The detection of inhibition and apoptotic rate of MRS5698 and cisplatin, and their combination, on Ishikawa and HEC-1A cells were performed by MTT assays and flow cytometry, respectively. The inhibition rates of MRS5698 on Ishikawa and HEC-1A cells were both significantly higher than the control groups ( P &lt; 0.05). MRS5698 produced a higher inhibitory effect on HEC-1A cells than on Ishikawa cells with IC50 values of 20.55 and 27.25 μg/mL, respectively. MRS5698 had a stronger inhibitory effect than cisplatin on HEC-1A cells. The Annexin V-FITC/propidium iodide assays demonstrated that the total rate of apoptosis of MRS5698 on HEC-1A cells was higher than that on Ishikawa cells. The results of MTT assay and cellular apoptosis showed that the combined use of MRS5698 and cisplatin produces dose-independent antagonistic effects. MRS5698 produced antitumor effects on both cell lines, which were better than that of cordycepin. However, the combined use of MRS5698 and cisplatin produced an antagonistic effect. A further in vivo study could be considered for investigating the antitumor effects of either MRS5698 monotherapy or MRS5698 in combination with other nonplatinum-based chemotherapeutic drugs in treating endometrial cancer.