Proliferation Of HEC-1A Cells Research Articles

The Role of Steroid Receptors in the Proliferation and Migration of Endometrial Adenocarcinoma HEC1A Cell Line

Objectives: Endometrial carcinoma is one of the most common gynecological cancers. It is generally divided into oestrogen-dependent type I, and oestrogen-independent type II. Although the expression of some steroid receptors has been documented in type II endometrial carcinoma, their roles in tumor progression have not been fully elucidated yet. Thus in this study, we aimed to examine the role of compounds acting on steroid receptors in type II, on HEC1A cultured cells.
 Methods: We tested the effect of mifepristone (the glucocorticoid and progesterone receptor blocker, 10-8M), bicalutamide (the androgen receptor blocker, 10-6M), G15 (the G-protein-coupled estrogen receptor-1 blocker, 10-7M) and PHTPP (2-Phenyl-3-(4-hydroxyphenyl)-5,7-bis (trifluoromethyl)-pyrazolo [1,5-a]pyrimidine, the estrogen receptor-β blocker, 10-7M), on proliferation. Proliferation was assessed by xCELLigence analysis system and migration was examined by using wound-healing model.
 Results: None of the drugs, at the used concentrations, have affected the proliferation of HEC1A cells. However, migration was significantly increased at the 24th and the 48th hour of mifepristone application (p

The role of potassium channels in the proliferation and migration of endometrial adenocarcinoma HEC1-A cells.

Endometrial cancer is the most common gynecological cancer in developed countries. Potassium channels, which have many types, are suggested to play a major role in cancer progression. However, their role in endometrial cancer has not been fully investigated. We aimed to demonstrate whether the ATP-sensitive potassium channel blocker glibenclamide, voltage-sensitive potassium channel blocker 4-aminopyridine, non-selective (voltage-sensitive and calcium-activated) potassium channels blocker tetraethylammonium and potassium chloride (KCl) have any effect on the proliferation and migration of HEC1-A cells. Proliferation and migration were evaluated by real-time cell analysis (xCELLigence system) and wound healing assays, respectively. Proliferation was reduced by glibenclamide (0.1 and 0.2mM, P < 0.05 and P < 0.01, respectively), 4-aminopyridine (10 and 20mM, P < 0.001) and tetraethylammonium (10 and 20mM, P < 0.01 and P < 0.001, respectively). However, KCl did not change the proliferation. Migration was reduced by glibenclamide (0.01, 0.1 and 0.2mM, P < 0.001, P < 0.001 and P < 0.01, respectively) and 4-aminopyridine (10 and 20mM, P < 0.05 and P < 0.01, respectively). Tetraethylammonium did not change migration. However, KCl reduced it (10, 25 and 50mM, P < 0.05, P < 0.01 and P < 0.01, respectively). Both proliferation and migration were reduced by glibenclamide and 4-aminopyridine. However, tetraethylammonium only reduced proliferation and KCl only reduced migration. Potassium channels have an important role in HEC1-A cell proliferation and migration and potassium channel blockers needs to be further investigated for their therapeutic effect in endometrial cancer.

Ginsenoside 20(S)-protopanaxadiol induces cell death in human endometrial cancer cells via apoptosis

Background20(S)-protopanaxadiol (20(S)-PPD), one of the aglycone derivatives of major ginsenosides, has been shown to have an anticancer activity toward a variety of cancers. This study was initiated with an attempt to evaluate its anti-cancer activity toward human endometrial cancer by cell and xenograft mouse models. MethodsHuman endometrial cancer (HEC)-1A cells were incubated with different 20(S)-PPD concentrations. 20(S)-PPD cytotoxicity was evaluated using MTT assay. Apoptosis was detected using the annexin V binding assay and cell cycle analysis. Cleaved poly (ADP-ribose) polymerase (PARP) and activated caspase-9 were assessed using western blotting. HEC-1A cell tumor xenografts in athymic mice were generated by inoculating HEC-1A cells into the flank of BALB/c female mice and explored to validate 20(S)-PPD anti-endometrial cancer toxicity. Results20(S)-PPD inhibited HEC-1A cell proliferation in a dose-dependent manner with an IC50 value of 3.5 μM at 24 h. HEC-1A cells morphologically changed after 20(S)-PPD treatment, bearing resemblance to Taxol-treated cells. Annexin V-positive cell percentages were 0%, 10.8%, and 58.1% in HEC-1A cells when treated with 0, 2.5, and 5 μM of 20(S)-PPD, respectively, for 24 h. 20(S)-PPD subcutaneously injected into the HEC-1A cell xenograft-bearing mice three times a week for 17 days manifested tumor growth inhibition by as much as 18% at a dose of 80 mg/kg, which sharply contrasted to controls that showed an approximately 2.4-fold tumor volume increase. These events paralleled caspase-9 activation and PARP cleavage. Conclusion20(S)-PPD inhibits endometrial cancer cell proliferation by inducing cell death via a caspase-mediated apoptosis pathway. Therefore, the 20(S)-PPD-like ginsenosides are endowed with ample structural information that could be utilized to develop other ginsenoside-based anticancer agents.

MiR-142 Suppresses Endometrial Cancer Proliferation In Vitro and In Vivo by Targeting Cyclin D1.

Endometrial cancer (EC), a prevalent gynecologic tumor, is a great threat to women. We aimed to explore miR-142's effects on EC and the relevant mechanisms. Cell proliferation was evaluated with MTT, cell counting, and colony formation assay. MRNA abundances of miR-142 and cyclin D1 (CCND1) were examined with quantitative real-time PCR. CCND1 protein level in cells was analyzed with Western blot. miR-142's downstream target was identified with targetscan and luciferase reporter assay. Nude mice were injected subcutaneously with Ishikawa (ISK) cells transfected with or without miR-142 mimics. Ki-67 and CCND1 expressions in tumors of xenograft mice were analyzed with immunohistochemical assay. miR-142 was expressed at a lower level in human EC tumor samples than matched normal tissues, and its mRNA level in EC patients without metastasis was higher than that in patients with metastatic EC. Additionally, low-level miR-142 was closely linked with the poor prognosis of EC patients. miR-142 restricted ISK and HEC-1A cell proliferation. Targetscan and luciferase reporter assay proved the target relationship between miR-142 and CCND1. Moreover, high-level CCND1 was positively correlated with the poor prognosis of EC patients. Besides, miR-142 mimics restricted tumor growth in ISK xenografted mice, as well as inhibited the expression of Ki67 and CCND1 in excised tumors. miR-142 restricted EC proliferation by targeting CCND1.

High throughput silencing identifies novel genes in endometrioid endometrial cancer

ObjectiveTo validate the gene expression profile obtained from the previous microarray analysis and to further study the biological functions of these genes in endometrial cancer. From our previous study, we identified 621 differentially expressed genes in laser-captured microdissected endometrioid endometrial cancer as compared to normal endometrial cells. Among these genes, 146 were significantly up-regulated in endometrial cancer. Materials and MethodsA total of 20 genes were selected from the list of up-regulated genes for the validation assay. The qPCR confirmed that 19 out of the 20 genes were up-regulated in endometrial cancer compared with normal endometrium. RNA interference (RNAi) was used to knockdown the expression of the upregulated genes in ECC-1 and HEC-1A endometrial cancer cell lines and its effect on proliferation, migration and invasion were examined. ResultsKnockdown of MIF, SOD2, HIF1A and SLC7A5 by RNAi significantly decreased the proliferation of ECC-1 cells (p < 0.05). Our results also showed that the knockdown of MIF, SOD2 and SLC7A5 by RNAi significantly decreased the proliferation and migration abilities of HEC-1A cells (p < 0.05). Moreover, the knockdown of SLC38A1 and HIF1A by RNAi resulted in a significant decrease in the proliferation of HEC1A cells (p < 0.05). ConclusionWe have identified the biological roles of SLC38A1, MIF, SOD2, HIF1A and SLC7A5 in endometrial cancer, which opens up the possibility of using the RNAi silencing approach to design therapeutic strategies for treatment of endometrial cancer.

Mammalian target of rapamycin inhibitor RAD001 sensitizes endometrial cancer cells to paclitaxel-induced apoptosis via the induction of autophagy.

The aim of the present study was to investigate the effects of the mammalian target of rapamycin (mTOR) inhibitor, RAD001, on the growth of human endometrial cancer cells. The effects of RAD001 on human endometrial cancer Ishikawa and HEC-1A cell proliferation were determined by MTT assay. Green fluorescent protein microtubule-associated protein 1 light chain 3α (GFP-LC3) protein aggregates were observed under a confocal microscope, and Ishikawa and HEC-1A cell apoptosis was detected using flow cytometry. The expression levels of LC3-I, LC3-II and mTOR proteins were detected by western blot analysis. The results showed that RAD001 effectively inhibited human endometrial cancer Ishikawa and HEC-1A cell proliferation via downregulation of AKT/mTOR phosphorylation. Moreover, RAD001 induced autophagic cell death and a higher sensitivity to paclitaxel-induced apoptosis. These results indicate that RAD001 could have therapeutic potential in human endometrial cancer with hyperactivated AKT/mTOR signaling.

Targeting Interleukin-11 Receptor-α Impairs Human Endometrial Cancer Cell Proliferation and Invasion In Vitro and Reduces Tumor Growth and Metastasis In Vivo.

Endometrial cancer contributes to significant morbidity and mortality in women with advanced stage or recurrent disease. IL11 is a cytokine that regulates cell cycle, invasion, and migration, all hallmarks of cancer. IL11 is elevated in endometrial tumors and uterine lavage fluid in women with endometrial cancer, and alters endometrial epithelial cancer cell adhesion and migration in vitro, but its role in endometrial tumorigenesis in vivo is unknown. We injected mice subcutaneously with human-derived Ishikawa or HEC1A endometrial epithelial cancer cells (ectopic), or HEC1A cells into the uterus (orthotopic) to develop endometrial cancer mouse models. Administration of anti-human IL11 receptor (R) α blocking antibody dramatically reduced HEC1A-derived tumor growth in both models and reduced peritoneal metastatic lesion spread in the orthotopic model, compared with IgG. Anti-human IL11Rα retained a well-differentiated, endometrial epithelial phenotype in the HEC1A ectopic mice, suggesting it prevented epithelial-to-mesenchymal transition. Blockade of mouse IL11Rα with anti-mouse IL11Rα antibody did not alter tumor growth, suggesting that cancer epithelial cell IL11 signaling is required for tumor progression. In vitro, anti-human IL11Rα antibody significantly reduced Ishikawa and HEC1A cell proliferation and invasion and promoted apoptosis. Anti-human, but not anti-mouse, IL11Rα antibody reduced STAT3, but not ERK, activation in HEC1A cells in vitro and in endometrial tumors in xenograft mice. We demonstrated that targeted blockade of endometrial cancer epithelial cell IL11 signaling reduced primary tumor growth and impaired metastasis in ectopic and orthotopic endometrial cancer models in vivo Our data suggest that therapeutically targeting IL11Rα could inhibit endometrial cancer growth and dissemination. Mol Cancer Ther; 15(4); 720-30. ©2016 AACR.

OPN induces FoxM1 expression and localization through ERK 1/2, AKT, and p38 signaling pathway in HEC-1A cells.

Mammalian embryo implantation is an extremely complex process and requires endometrial receptivity. In order to establish this receptivity, sequential proliferation and differentiation during the menstrual cycle is necessary. Forkhead box M1 (FoxM1) is described as a major oncogenic transcription factor in tumor initiation, promotion and progression. According to these functions, we believe that FoxM1 should also play an essential role in embryo implantation. Osteopontin (OPN), an adhesion molecule, has been studied extensively in reproduction. In this study, we observed the expression and distribution of FoxM1 during the proliferative-phase and secretory-phase human endometrium and the pre-implantation mouse uterus firstly. Then we observed the relationship between OPN and FoxM1. Our results showed that FoxM1 was mainly distributed in glandular epithelium. OPN increased the expression of FoxM1 in the human uterine epithelial cell line HEC-1A cells in a time- and concentration-dependent manner. OPN regulates FoxM1 to influence HEC-1A cell proliferation through extracellular regulated protein kinases (ERK 1/2), protein kinase B (PKB, AKT), and the p38 mitogen activated protein kinases (p38MAPK, p38) signaling pathway. Inhibition of ERK 1/2, AKT and p38 suppressed OPN-induced FoxM1 expression and location. Our data indicate that FoxM1 might be regulated by OPN to influence endometrial proliferation to establish endometrial receptivity.

Icaritin Causes Sustained ERK1/2 Activation and Induces Apoptosis in Human Endometrial Cancer Cells

Icaritin, a compound from Epimedium Genus, has selective estrogen receptor (ER) modulating activities, and posses anti-tumor activity. Here, we examined icaritin effect on cell growth of human endometrial cancer Hec1A cells and found that icaritin potently inhibited proliferation of Hec1A cells. Icaritin-inhibited cell growth was associated with increased levels of p21 and p27 expression and reduced cyclinD1 and cdk 4 expression. Icaritin also induced cell apoptosis accompanied by activation of caspases as evidenced by the cleavage of endogenous substrate Poly (ADP-ribose) polymerase (PARP) and cytochrome c release, which was abrogated by pretreatment with the pan-caspase inhibitor z-VAD-fmk. Icaritin treatment also induced expression of pro-apoptotic protein Bax with a concomitant decrease of Bcl-2 expression. Furthermore, icaritin induced sustained phosphorylation of extracellular signal-regulated kinase1/2 (the MAPK/ ERK1/2) in Hec1A cells and U0126, a specific MAP kinase kinase (MEK1/2) inhibitor, blocked the ERK1/2 activation by icaritin and abolished the icaritin-induced growth inhibition and apoptosis. Our results demonstrated that icaritin induced sustained ERK 1/2 activation and inhibited growth of endometrial cancer Hec1A cells, and provided a rational for preclinical and clinical evaluation of icaritin for endometrial cancer therapy.

Lysophosphatidic acid (LPA) effects on endometrial carcinoma in vitro proliferation, invasion, and matrix metalloproteinase activity

Objectives Lysophosphatidic acid (LPA) has potent growth-regulatory effect in many cell types and has been linked to the in vivo tumor growth and metastasis in several malignancies. The goal of this study was to assess the regulation of (EC) microenvironment by LPA through the examination of its effect on cell proliferation, migration, invasion, uPA activity, and matrix metalloproteinase (MMP) secretion/activation. Methods All experiments were performed in vitro using an EC cell line, HEC-1A. Cell proliferation was determined using the Promega MTS proliferation assay following 48 h of exposures to different concentrations of LPA (0.1, 1.0 and 10.0 μM). Cell invasion was assessed using a modified Boyden chamber assay with collagen I coated on the membrane. HEC-1A motility was examined by Boyden chamber migration assay as well as the scratch wound closure assay on type I collagen. MMP secretion/activation in HEC-1A conditioned medium was detected by gelatin zymography. MMP-7 mRNA expression was determined using real-time PCR. uPA activity was measured using a coupled colorimetric assay. Results LPA, at the concentrations of 0.1 and 1.0 μM, significantly induced the proliferation of HEC-1A cells ( p < 0.01). At 10 μM, LPA- induced HEC-1A proliferation to a less extent and showed no significant effect on HEC-1A invasion and migration ( p > 0.05). Gelatin zymogram showed that HEC-1A cells secreted high levels of MMP-7, while MMP-2 and MMP-9 are barely detectable. In addition, LPA significantly enhanced uPA activity in HEC-1A conditioned medium in a concentration-dependent manner. Conclusions LPA is a potent modulator of cellular proliferation and invasion for EC cells. It also has the capacity to stimulate the secretion/activity of uPA and MMP-7. Those results suggest that LPA is a bioactive modulator of EC microenvironment and may have a distinct regulation mechanism as observed in epithelial ovarian cancer.

The effect of rosiglitazone, peroxisome proliferator-activated receptor gamma agonist on human telomerase reverse transcriptase expression in HEC-1A cell

OBJECTIVE: The objective of this study was to investigate the effect of rosiglitazone, PPAR gamma agonist on hTERT(human telomerase reverse transcriptase) expression, ER(estrogen receptor) translocation into nucleus and ERK phosphorylation in HEC-1A cells.DESIGN: Telomerase, the specialized reverse transcriptase enzyme that elongates telomeres and synthesizes telomeric DNA, is increased in most cancer cells. Telomere shortening with each cell division, leading to limitless replication potential. PPAR gamma can suppress the cell growth in many cancer cells through various signal pathway. Therefore, we evaluated whether PPAR gamma has influence on hTERT expression.MATERIALS AND METHODS: HEC-1A cell was obtained from the American Type Culture Collection. After treatment of rosiglitazone, PPAR gamma agonist, hTERT expression, ERK phosphorylation and ER nuclear translocation were examined by western blotting in HEC-1A.RESULTS: The expression of hTERT was decreased in human endometrial cancer cell by treatment with rosiglitazone, PPAR gamma agonist. Also, decrements of ERK phosphorylation and ER beta nuclear translocation were observed.CONCLUSIONS: These results suggest that PPAR gamma plays an important role in the HEC-1A cell proliferation by hTERT expression. OBJECTIVE: The objective of this study was to investigate the effect of rosiglitazone, PPAR gamma agonist on hTERT(human telomerase reverse transcriptase) expression, ER(estrogen receptor) translocation into nucleus and ERK phosphorylation in HEC-1A cells. DESIGN: Telomerase, the specialized reverse transcriptase enzyme that elongates telomeres and synthesizes telomeric DNA, is increased in most cancer cells. Telomere shortening with each cell division, leading to limitless replication potential. PPAR gamma can suppress the cell growth in many cancer cells through various signal pathway. Therefore, we evaluated whether PPAR gamma has influence on hTERT expression. MATERIALS AND METHODS: HEC-1A cell was obtained from the American Type Culture Collection. After treatment of rosiglitazone, PPAR gamma agonist, hTERT expression, ERK phosphorylation and ER nuclear translocation were examined by western blotting in HEC-1A. RESULTS: The expression of hTERT was decreased in human endometrial cancer cell by treatment with rosiglitazone, PPAR gamma agonist. Also, decrements of ERK phosphorylation and ER beta nuclear translocation were observed. CONCLUSIONS: These results suggest that PPAR gamma plays an important role in the HEC-1A cell proliferation by hTERT expression.

Ligand-dependent and -independent effects of splice variant 1 of growth hormone-releasing hormone receptor.

Existing evidence indicates that, in addition to its neuroendocrine action, growth hormone-releasing hormone (GHRH) acts directly on several nonpituitary tissues, especially neoplasms, and stimulates cell proliferation. We have recently reported that a splice variant of the receptor (SV1) is expressed in various normal tissues and particularly in tumor tissues, producing mitogenic effects on GHRH binding. By using HEC-1A human endometrial carcinoma cells, which express endogenous SV1, we show that, in addition to its ability to mediate the mitogenic effects of GHRH, SV1 also possesses relatively high intrinsic, ligand-independent activity. By using an antisense RNA-based approach we found that SV1 ablation reduces the efficacy of colony formation and the rate of cell proliferation of HEC-1A cells in the absence of exogenous GHRH, and decreases their sensitivity to GHRH when the neurohormone is added to the culture media. This ligand-independent stimulation of cell proliferation appears to be a characteristic property of the truncated form of the receptor, because the expression of SV1 and not of the full-length GHRH receptor stimulated the proliferation of 3T3 fibroblasts in the absence of exogenous GHRH, whereas both forms mediated the proliferative effects of GHRH. Evaluation of 21 specimens of human primary endometrial carcinoma for expression of SV1 by immunohistochemistry indicated that in contrast to the GHRH receptor, which is absent, SV1 is expressed in approximately 43% of the specimens. These findings indicate that SV1 can operate in a ligand-independent as well as a ligand-dependent manner. The overexpression of this form of GHRH receptor may be associated with carcinogenesis.

Differential Inhibitory Effects on Human Endometrial Carcinoma Cell Growth of Luteinizing Hormone-Releasing Hormone Analogues

In addition to its function as a key hormone in the regulation of the pituitary–gonadal axis, luteinizing hormone-releasing hormone (LHRH) probably also affects various extrapituitary tissues. LHRH binding sites andin vitroantiproliferative effects of LHRH analogues have been reported in human endometrial cancer. The effects of the LHRH agonist leuproreline and LHRH antagonist antide were studied on the cell growth, DNA synthesis, and cell cycle distribution of the human endometrial cancer cell lines HEC-1A and HEC-1B by the sulforhodamine B (SRB) method, [3H]thymidine assay incorporation, and propidium iodide DNA staining, respectively. In the presence of 1.0–100 μM leuproreline the proliferation of HEC-1A cells was significantly reduced as early as 3 days after drug exposure, with a minimum growth value of 69.9 ± 3.6% (mean ± SE) at the highest concentration tested (100 μM). Similar antiproliferative effects were obtained following a 6-day treatment with the LHRH antagonist antide. Also, inhibitory effects on [3H]thymidine incorporation into the DNA of the HEC-1A cell line were noted after a 6-day exposure to both LHRH analogues, in the above-mentioned concentration range. Cell cycle analysis of HEC-1A cells cultured in the presence of 10 μM leuproreline and antide showed a slight accumulation of cells in the G0/G1phase, while the proportions of cells in the S and G2/M phases concomitantly decreased. No significant effects on proliferation, DNA synthesis, and cell cycle distribution were observed in HEC-1B cells with either leuproreline or antide (up to 100 and 10 μM, respectively) after a 6-day exposure. Both Northern blot analysis and reverse transcription polymerase chain reaction failed to detect expression of mRNA for the LHRH receptor in both HEC-1A and HEC-1B cell lines. In addition, the LHRH analogues did not affect the intracellular free calcium concentration, indicating that the classic signal transduction for LHRH is absent or impaired in HEC-1A cells. The observed direct inhibitory actions on HEC-1A cells support the concept that the two LHRH analogues may exert biological effects via cellular effectors distinct from the “classic” LHRH receptor. Although the mechanism by which these direct actions are produced is still obscure, these results might help to establish the basis for new approaches to the therapy of endometrial cancer.