HEC-50B Cells Research Articles

Mitochondrial Dynamics of Bcl-2 Family Proteins during 17-β-Estradiol-Induced Apoptosis Correlate with the Malignancy of Endometrial Cancer Cells.

Excessive fat intake leads to an increase in cholesterol. Overexposure to estrogen derived from cholesterol is known to contribute to the malignancy of endometrial adenocarcinomas. However, it is not well understood the relationship between the exposure to estrogen and the malignancy of endometrial adenocarcinomas. We investigated how estrogen affected the malignancy of endometrial cancer cells, specifically HEC1 cells (a moderately differentiated adenocarcinoma) and HEC50B cells (a poorly differentiated adenocarcinoma). Cell viability was decreased by exogenous 17-β-estradiol (E2) in a concentration-dependent manner. E2 disturbed the mitochondrial membrane potentials by changing the localization of the B-cell lymphoma 2 (Bcl-2) family protein; however, there were significant differences in the localization of Bcl-2 family proteins between HEC1 and HEC50B cells. In HEC1 cells, E2 increased the expression of B-cell lymphoma-extra large (Bcl-XL) and the Bcl-2-associated X protein (Bax) and decreased Bcl-2 and Bcl-2-associated death promoter (Bad) expression on the outer mitochondrial membrane. Conversely, E2 increased the expression of Bad and Bax, and it decreased Bcl-2 and Bcl-XL expressions on the outer mitochondrial membrane in HEC50B cells. The disturbance of the mitochondrial membrane potential led to the release of cytochrome c from the mitochondria to the cytosolic space followed by activating caspase-9. After that, caspase-3 was activated and induced apoptosis. These results suggested that the localization of the Bcl-2 family protein observed under E2-induced apoptosis is related to the malignancy of endometrial cancer cells. We hope that the dynamics of Bcl-2 family proteins such as Bcl-XL and Bad will be used to diagnose malignant endometrial adenocarcinomas.

LIM1 contributes to the malignant potential of endometrial cancer.

The incidence of endometrial cancer (EC) has been increasing worldwide. However, because there are limited chemotherapeutic options for the treatment of EC, the prognosis of advanced-stage EC is poor. Gene expression profile datasets for EC cases registered in The Cancer Genome Atlas (TCGA) was reanalyzed. Highly expressed genes in advanced-stage EC (110 cases) compared with early-stage EC (255 cases) were extracted and Gene Ontology (GO) enrichment analysis was performed. Among the enriched genes, Kaplan-Meier (KM) plotter analysis was performed. Candidate genes expression was analyzed in HEC50B cells and Ishikawa cells by RT-qPCR. In HEC50B cells, LIM homeobox1 (LIM1) was knocked down (KD) and cell proliferation, migration, and invasion ability of the cells were evaluated. Xenografts were generated using LIM1-KD cells and tumor growth was evaluated. Ingenuity Pathway Analysis (IPA) of RNA-seq data using LIM-KD cells was performed. Expression of phospho-CREB and CREB-related proteins were evaluated in LIM1-KD cells by western blotting and in xenograft tissue by immunofluorescent staining. Two different CREB inhibitors were treated in HEC50B and cell proliferation was evaluated by MTT assay. Reanalysis of TCGA followed by GO enrichment analysis revealed that homeobox genes were highly expressed in advanced-stage EC. Among the identified genes, KM plotter analysis showed that high LIM1 expression was associated with a significantly poorer prognosis in EC. Additionally, LIM1 expression was significantly higher in high-grade EC cell lines, HEC50B cells than Ishikawa cells. Knockdown of LIM1 showed reduced cell proliferation, migration and invasion in HEC50B cells. Xenograft experiments revealed that tumor growth was significantly suppressed in LIM1-KD cells. IPA of RNA-seq data using LIM-KD cells predicted that the mRNA expression of CREB signaling-related genes was suppressed. Indeed, phosphorylation of CREB was decreased in LIM1-KD cells and LIM1-KD cells derived tumors. HEC50B cells treated by CREB inhibitors showed suppression of cell proliferation. Collectively, these results suggested that high LIM1 expression contributed to tumor growth via CREB signaling in EC. Inhibition of LIM1 or its downstream molecules would be new therapeutic strategies for EC.

Expression of cell adhesion molecule 1 in human and murine endometrial glandular cells and its increase during the proliferative phase by estrogen and cell density

AimsCell adhesion molecule 1 (CADM1) mediates interepithelial adhesion and is upregulated in crowded epithelial monolayers. This study aimed to examine CADM1 expression in the human endometrium of proliferative and secretory phases, and its transcriptional regulation in terms of estrogen stimuli and higher cellularity. Main methodsCADM1 immunohistochemistry was conducted on endometrial tissues from women in their 40s and adult mice subcutaneously injected with estradiol following ovariectomy. Dual-luciferase reporter assays were conducted using human endometrial HEC-50B and HEC-1B cells and reporter plasmids harboring the human CADM1 3.4-kb promoter and its deleted and mutated forms. Cells were transfected with estrogen receptor α cDNA and reporter plasmids, and treated with estradiol before luciferase activity measurement. Key findingsImmunohistochemistry revealed that CADM1 was clearly expressed on the lateral membranes of the simple columnar glandular cells in the proliferative phase, but not in the secretory phase, from both women and the mouse model. The glandular cell density increased two-fold in the proliferative phase. Reporter assays identified three Sp1-binding sites as estradiol-responsive elements in the proximal region (from −223 to −84) of the transcription start site (+1) in HEC-50B cells. When the cell culture was started at eight-fold higher cell density, the CADM1 3.4-kb promoter was transactivated at a two-fold higher level in HEC-50B cells. This cell density effect was not detected for the CADM1 2.3-kb or 1.6-kb promoter. SignificanceTwo (proximal and distal) promoter regions are suggested to function additively to transactivate CADM1 in endometrial glandular cells that crowd in the proliferative phase.

Proliferation of poorly differentiated endometrial cancer cells through autocrine activation of FGF receptor and HES1 expression.

Patients with poorly differentiated endometrial cancer show poor prognosis, and effective molecular target-based therapies are needed. Endometrial cancer cells proliferate depending on the activation of HES1 (hairy and enhancer of split-1), which is induced by several pathways, such as the Notch and fibroblast growth factor receptor (FGFR) signaling pathways. In addition, aberrant, ligand-free activation of the FGFR signaling pathway resulting from mutations in FGFR2 was also reported in endometrial cancer. However, a clinical trial showed that there was no difference in the effectiveness of FGFR inhibitors between patients with and without the FGFR2 mutation, suggesting a presence of another signaling pathway for the FGFR activation. Here, we investigated the signaling pathway regulating the expression of HES1 and proliferation of poorly and well-differentiated endometrial cancer cell lines Ishikawa and HEC-50B, respectively. Whereas Ishikawa cells proliferated and expressed HES1 in a Notch signaling-dependent manner, Notch signaling was not involved in HES1 and proliferation of HEC-50B cells. The FGFR inhibitor, NVP-BGJ398, decreased HES1 expression and proliferation of HEC-50B cells; however, HEC50B cells had no mutations in the FGFR2 gene. Instead, HEC-50B cells highly expressed ligands for FGFR2, suggesting that FGFR2 is activated by an autocrine manner, not by ligand-free activation. This autocrine pathway activated Akt downstream of FGFR for cell proliferation. Our findings suggest the usefulness of HES1 as a marker for the proliferation signaling and that FGFR inhibitor may be effective for poorly differentiated endometrial cancers that harbor wild-type FGFR.

FOXP1 forkhead transcription factor is associated with the pathogenesis of endometrial cancer

Endometrial cancers are mostly estrogen-dependent. FOXP1 is a P subfamily of forkhead box (FOX), and known as an estrogen-responsive transcription factor. The aims of this study were to examine histological location of FOXP1 in normal and malignant endometrium, and to investigate a possible association between FOXP1 and other factors considered to be involved in pathogenesis of endometrial cancer. The levels of FOXP1, estrogen receptor (ER)α, and ERβ expression were examined immunohistochemically in normal and malignant endometrium obtained from 75 women (8 normal, 8 atypical endometrial hyperplasia, and 59 endometrial cancers from grade 1 to 3). The effects of estrogen on ERα, FOXP1, KRAS, and PTEN expression were analyzed in telomerase-immortalized human endometrial stromal cells (T HESCs) by Western blotting. Western blotting was also used to examine the effect of FOXP1 plasmid DNA or siRNA transfection on KRAS and PTEN expression in Ishikawa cells (well differentiated endometrioid adenocarcinoma), HEC-50B cells (poorly differentiated endometrioid adenocarcinoma), and T HESCs, respectively. FOXP1 was expressed in normal and malignant endometrium, but the rate of expression was different depending upon menstrual cycle and pathological grade of malignancy. FOXP1 expression in nucleus and cytoplasm of grade 3 endometrioid cancers was significantly lower than that of grade 1 and 2 ones. Estradiol increased levels of FOXP1 and KRAS expression in a dose- and time-dependent manner in T HESCs cells, and FOXP1 transfection or knockdown led to increase or decrease of KRAS expression but not PTEN. KRAS expression level was significantly related to FOXP1 and ERα levels in cancer tissues. Estradiol did not affect KRAS expression in T HESCs cells transfected with FOXP1 siRNA. These results suggest that FOXP1 is involved in estrogen dependent endometrial cancers through KRAS pathway.

MiR-205 promotes epithelial-mesenchymal transition by targeting AKT signaling in endometrial cancer cells.

AKT signaling regulates multiple biological processes and expresses in various cancers. miR-205 plays complex roles in tumorigenesis and tumor progression by acting either as a tumor suppressor or an oncogene depending on the tumor type. Here we describe the molecular mechanism of miR-205 regulating epithelial-mesenchymal transition by activation of AKT signaling in endometrial cancer cells HEC-50B and HEC-1-A. The proliferation of HEC-50B cells transfected with miR-205 mimic was assessed by WST-1 assay. The migration and invasion were evaluated by BD transwell migration and matrigel invasion assays. The EMT markers were detected by Western blot. We found that miR-205 increased the proliferation in HEC-50B cells. The migration and invasion of HEC-50B cells and HEC-1-A cells were enhanced by miR-205. When HEC-50B cells and HEC-1-A cells were treated with anti-miR-205 inhibitor, the migration and invasion were decreased as compared with the negative control. The overexpression of miR-205 inhibited E-cadherin expression and promoted Snail expression by activation of AKT and downregulation of glycogen synthase kinase 3β. However, after the HEC-50B cells and HEC-1-A cells were treated with anti-miR-205 inhibitor, E-cadherin expression was increased and Snail protein level was decreased by inhibition of AKT expression. Our data strongly suggest that miR-205 plays an important role in endometrial cancer migration and invasion by targeting the AKT pathway. Our data highlight miR-205 as a potential molecular target for endometrial cancer treatment.

MicroRNA 31 functions as an endometrial cancer oncogene by suppressing Hippo tumor suppressor pathway.

BackgroundWe aimed to investigate whether MIR31 is an oncogene in human endometrial cancer and identify the target molecules associated with the malignant phenotype.MethodsWe investigated the growth potentials of MIR31-overexpressing HEC-50B cells in vitro and in vivo. In order to identify the target molecule of MIR31, a luciferase reporter assay was performed, and the corresponding downstream signaling pathway was examined using immunohistochemistry of human endometrial cancer tissues. We also investigated the MIR31 expression in 34 patients according to the postoperative risk of recurrence.ResultsThe overexpression of MIR31 significantly promoted anchorage-independent growth in vitro and significantly increased the tumor forming potential in vivo. MIR31 significantly suppressed the luciferase activity of mRNA combined with the LATS2 3’-UTR and consequently promoted the translocation of YAP1, a key molecule in the Hippo pathway, into the nucleus. Meanwhile, the nuclear localization of YAP1 increased the transcription of CCND1. Furthermore, the expression levels of MIR31 were significantly increased (10.7-fold) in the patients (n = 27) with a high risk of recurrence compared to that observed in the low-risk patients (n = 7), and this higher expression correlated with a poor survival.ConclusionsMIR31 functions as an oncogene in endometrial cancer by repressing the Hippo pathway. MIR31 is a potential new molecular marker for predicting the risk of recurrence and prognosis of endometrial cancer.

Utilization of genomic signatures to identify high‐efficacy candidate drugs for chemorefractory endometrial cancers

Endometrial cancer, one of the most common gynecologic malignancies, is increasing in Japan, nearly doubling over the last decade. High-grade disease patients are often resistant to conventional chemotherapy with platinum agents; therefore, discovery of efficacious new drugs in this setting is required to benefit chemorefractory cases. The 50% growth-inhibitory (GI50) concentration of 27 clinically relevant drugs was measured in the NCI60 panel of cell lines. Gene expression data were analyzed using Bayesian binary regression, to first generate a response signature for each drug and then to calculate individual susceptibility scores using in vivo endometrial cancer data (GSE2109; http://www.ncbi.nlm.nih.gov/geo) and in vitro data (GSE25458), as well as to identify candidate drugs for chemorefractory cases. Using these candidates, cell proliferation, apoptosis and caspase assays were performed in vitro. The tumor growth-inhibitory effect of the candidate was also assessed in vivo using nude mice. Through microarray analysis, fludarabine and temsirolimus showed higher susceptibility scores in high-grade cases compared to cisplatin, doxorubicin and paclitaxel. Fludarabine significantly inhibited cell proliferation and increased apoptosis in the cisplatin-resistant endometrial cancer cell line, HEC1A, relative to HEC50B (p < 0.001). Fludarabine treatment also enhanced caspase-3/7 activity in HEC1A relative to HEC50B cells (p < 0.001), and inhibited the growth of HEC1A xenograft tumors relative to cisplatin (p < 0.05). These results support that identification and use of genomic signatures can lead to identification of new therapeutic candidates that may prove beneficial to chemoresistant cases. Fludarabine may be useful in targeting high-grade, chemorefractory endometrial cancer.

Downregulation of vasohibin-2, a novel angiogenesis regulator, suppresses tumor growth by inhibiting angiogenesis in endometrial cancer cells

The vasohibin-2 (VASH2) gene was originally found to be expressed in infiltrating mononuclear cells of a mouse model of hypoxia-induced subcutaneous angiogenesis. These cells are mobilized from bone marrow to promote angiogenesis. Recently, VASH2 has been demonstrated to be expressed in several types of cancer in which it promotes tumor development through angiogenesis. However, its role in endometrial cancer remains unknown. Using quantitative reverse transcription-polymerase chain reaction (RT-PCR), we found that VASH2 was overexpressed in several human endometrial cancer cell lines, including the HEC50B cell line, which we used to further examine the role of VASH2. Although knockdown of VASH2 with stable transfection of shRNA had little effect on the proliferation of HEC50B cells in vitro, knockdown in an in vivo murine xenograft model inhibited tumor growth by decreasing tumor angiogenesis. In addition, the supernatant from HEC50B cells that expressed VASH2 significantly promoted the proliferation of human umbilical vein endothelial cells. By contrast, knockdown of VASH2 significantly attenuated the proliferative effect. These results indicate that VASH2 contributes to the development of endometrial cancer by promoting angiogenesis through a paracrine mode of action. Consequently, VASH2 may be considered to be a novel molecular target for endometrial cancer therapy.

Effect of p53 gene transfection on vascular endothelial growth factor expression in endometrial cancer cells

It has been reported that tumor suppressor gene p53 regulates vascular endothelial growth factor (VEGF) expression, but the relation between them in endometrial carcinoma remains unclear. We investigated VEGF expression in 11 endometrial carcinoma cell lines and the effect of p53 gene transfection on VEGF expression in the p53-mutated endometrial carcinoma cell line, HEC-50B. Immunoblotting for detecting VEGF, p53, and β-actin was performed. Wild type p53 gene was transfected using the SuperFect method. The mean VEGF value of 0.8 ± 0.3 ( n = 6) in p53 wild-type group was significantly lower than the 1.6 ± 0.8 ( n = 5) that was found in the p53 mutant group ( P < 0.05). Levels of VEGF in the culture medium were measured by enzyme immunoassay (EIA). VEGF levels in the p53 gene-transfected HEC-50B cells and the conditioned medium were decreased at 48 h after p53 gene transfection. VEGF expression was downregulated by p53 in endometrial carcinoma cells.

VEGF expression and its reguration by p53 gene transfection in endometrial carcinoma cells.

Vascular endothelial growth factor (VEGF) that activates endothelial cell growth induces angiogenesis, which is indispensable to tumor igenesis and tumor progression. On the other hand, tumor suppressor gene p53 has been considered to regulate VEGF expression, but the detailed relationship between them remains unclear. In this study, we aimed to study VEGF expression in endometrial carcinoma cells and the effect of p53 gene transfection on VEGF expression using p53-mutated endometrial carcinoma cell line, HEC-50B. Immunoblotting for detecting VEGF protein, p53 protein and beta-actin was performed using 11 endometrial carcinoma cell lines. Levels of VEGF in the cultured media were measured by Enzyme immunoassay(EIA). Transfection of wild p53 gene was carried out by SuperFect method in HEC-50B cells, which had mutant p53 gene and did not express p53 protein. The results of immunoblotting were analyzed by NIH image and expressed as values. The results of EIA were expressed as the relative value. The VEGF value was 0.8 +/- 0.3 (n = 6) in p53-wild group, whereas in p53-mutant group it was 1.6 +/- 0.8 (n = 5). VEGF expression was correlated significantly with p53 status (P < 0.05). VEGF levels in p53 gene-transfected cells and the conditioned medium were decreased in 48 hours after p53 gene transfection. VEGF expression was down-regulated by p53 in endometrial carcinoma cells.