Endometrial Carcinoma Ishikawa Cells Research Articles

The Effects of Lithium, Metformin and Everolimus Substances on Cell Growth in 2D and 3D Ishikawa Endometrial Carcinoma Cell Culture

Purpose: Our aim is to study the effects of the single and combined treatments of Everolimus, Metformin, and Lithium Chloride in two-dimensional (2D, monolayer) and three-dimensional (3D, spheroid) cell cultures of Ishikawa cells, which comprise the endometrial cancer cell line. Materials and methods: As part of the study, the effects of single and combined forms of Everolimus, Metformin, and Lithium Chloride were determined on cell viability, invasion, colony formation and apoptosis, and PI3K/AKT/mTOR pathway. Cell viability was assessed using XTT assay. CASP3, CASP8, CASP9, FASL, FADD, TNF, TRADD, BAX, P53, PI3KCA, PI3KCB, PTEN, MTOR, AKT1 genes were evaluated with RT-PCR, apoptosis was evaluated by flow cytometry and 3D spheroid results were evaluated with invert microscope analysis. Results: Everolimus, metformin, and lithium's IC50 levels were found at 48 hours to be 37.46 nM, 48.59 mM, and 100 µM, respectively. It was determined that the invasive capacities of Ishikawa cells in treatment groups, as well as cell colony formation were significantly reduced. In addition, Ishikawa spheroid cells were significantly suppressed compared with the control groups. RT-PCR results revealed that substances and their combinations affect genes associated with PI3K/AKT/mTOR pathway and apoptosis. Flow cytometry results showed notably increased apoptosis by single and combined treatments. Conclusion: As a result, the single and combination forms of everolimus, metformin, and lithium have reduced cell proliferation, induced apoptosis, and decreased mTOR activation through various mechanisms in Ishikawa cells.

Explore the anti-inflammation mechanism of safflower total flavonoids in the treatment of endometritis based on celluar transcriptomics.

Safflower is a traditional Chinese medicine for the treatment of gynecological diseases and its flavonoids have potential anti-inflammatory effects. The purpose is to explore the possible effects of safflower total flavonoids (STF) on lipopolysaccharide (LPS)-induced inflammatory damage of Ishikawa cells. In this study, LPS-induced endometrial carcinoma Ishikawa cells were used to establish an inflammatory injury model. The effective concentration of STF was screened by CCK-8 and enzyme-linked immunosorbent assay. The apoptosis of damaged Ishikawa cells was detected by flow cytometry. The contents of caspase11 and caspase 3 in Ishikawa cells were observed by fluorescence imaging. Western blot and RT-qPCR were used to detect the expression of related proteins and mRNA in damaged Ishikawa cells, and the possible mechanism of safflower flavonoids against LPS-induced endometrial carcinoma Ishikawa cells was analyzed by cell transcriptomics. The STF-reduced tumor necrosis factor α, interleukin-1β, and interleukin-6 expression level; the expression level of the proteins ASK1, Caspase3, and Caspase11 was also significantly decreased, and the proteins ERα, p-PI3K, and p-AKT were significantly increased. The transcriptome results showed that the PI3K-Akt signal pathway may be the main signal pathway for the STF. The STF could regulate the PI3K/AKT signal pathway to treat the inflammatory injury of Ishikawa cells.

Apigenin, a natural flavonoid, promotes autophagy and ferroptosis in human endometrial carcinoma Ishikawa cells in vitro and in vivo

Apigenin, a natural flavonoid has been reported against a variety of cancer types. However, it is unclear whether apigenin can promote autophagy and ferroptosis in Ishikawa cells. There are few reports on the mechanism of apigenin on autophagy and ferroptosis of endometrial cancer Ishikawa cells. We found that iron accumulation, lipid peroxidation, glutathione consumption, p62, HMOX1, and ferritin were increased, while, solute carrier family 7 member 11 and glutathione peroxidase 4 were decreased. Ferrostatin-1, an iron-death inhibitor could reverse the effects of apigenin in Ishikawa cells. On the other hand, apigenin could promote autophagy via up-regulating Beclin 1, ULK1, ATG5, ATG13, and LC3B and down-regulating AMPK, mTOR, P70S6K, and ATG4. Furthermore, apigenin could inhibit tumor tissue proliferation and restrict tumor growth via ferroptosis in vivo.

Integrated miRNA and mRNA omics reveal dioscin suppresses migration and invasion via MEK/ERK and JNK signaling pathways in human endometrial carcinoma in vivo and in vitro

Ethnopharmacological relevancePolygonatum sibiricum Redouté (PS, also called Huangjing in traditional Chinese medicine), is a perennial herb as homology of medicine and food. According to the traditional Chinese medicine theory “Special Records of Famous Doctors”, its functions include invigorating qi and nourishing yin, tonifying spleen and kidney. Traditionally, qi and blood therapy has been believed as most applicable to the treatment of uterine disease. The current research has focused on the effect and mechanism of dioscin, the main active component of PS, on Endometrial carcinoma (EC). Aim of the studyTo study the efficacy of dioscin on proliferation and migration of Endometrial carcinoma cell line, we conducted experiments by using xenograft model and Ishikawa cells, and explored the potential molecular mechanism. Materials and methodsmRNA and miRNA omics techniques were employed to investigate the regulatory mechanism of dioscin on EC Ishikawa cells. Based on in vivo and in vitro experiments, cell clone formation, cell scratching, Transwell, H&E staining, immunohistochemistry, q-PCR, and Western blot techniques were used to determine the molecular effects and mechanisms of dioscin on cell migration. ResultsIntegrated miRNA and mRNA omics data showed that 513 significantly different genes marked enrichment in MAPK signaling pathway. The in vivo data showed that dioscin (24 mg/kg) significantly inhibited tumor growth. The in vitro proliferation and invasiveness of dioscin on Ishikawa cells showed that dioscin could significantly decrease the colony numbers, and suppress the Ishikawa cell wound healing, migration and invasion. Molecular data revealed that dioscin decreased the MMP2 and MMP9 expression in vitro and in vivo. The p-MEK, p-ERK, and p-JNK expression levels were also confirmed to be significantly reduced. Key regulators in the MAPK signaling pathway were further validated in xenograft tumors. ConclusionOur data indicated that dioscin inhibited Ishikawa cell migration and invasion mediated through MEK/ERK and JNK signaling. More importantly, screened hub miRNAs and genes can be regarded as potential molecular targets for future EC treatment.

Isorhamnetin Induces Apoptosis and Suppresses Metastasis of Human Endometrial Carcinoma Ishikawa Cells via Endoplasmic Reticulum Stress Promotion and Matrix Metalloproteinase-2/9 Inhibition In Vitro and In Vivo.

Endometrial cancer (EC) is a very common female cancer which has attracted more and more attention. According to the individual patient's condition, the current treatment of EC patients is mainly based on surgery, which is supplemented by chemotherapy, radiotherapy, and endocrine intervention. However, these existing treatment strategies also have some inevitable limitations. Therefore, it is particularly important to find an active ingredient with low toxicity and a high safety profile against EC. Isorhamnetin is a flavonoid known to be present in a variety of plants, such as sea buckthorn, dry willow, and wolfberry. In recent years, the anti-tumor effects of isorhamnetin have been reported. In our study, isorhamnetin was shown to induce apoptosis in Ishikawa cells by inducing the endogenous mitochondrial apoptotic pathway and exogenous death receptor pathway, promoting the endoplasmic reticulum stress-related pathway, and activating the corresponding markers of UPR response. In addition, isorhamnetin affected the expression of MMP2 and MMP9-related proteins in vitro and in vivo and eventually repressed metastasis. Therefore, isorhamnetin can be used as a promising medicinal material for the treatment of EC.

Apigenin inhibits migration and induces apoptosis of human endometrial carcinoma Ishikawa cells via PI3K-AKT-GSK-3β pathway and endoplasmic reticulum stress

Several experimental and biological studies have emphasized the tumor suppression efficacy and low toxicity of Apigenin (API); however, its exact underlying mechanism on human endometrial carcinoma Ishikawa cell line (EC) is still unknown. We found that API could inhibit the proliferation of Ishikawa cells at IC50 of 45.55 μM, arrest the cell cycle at G2/M phase, induce apoptosis by inhibiting Bcl-xl and increasing Bax, Bak and Caspases. Further, API could induce apoptosis by activating the endoplasmic reticulum (ER) stress pathway by increasing the Ca2+, ATF4, and CHOP. It could impede cell migration and invasion through PI3K-AKT-GSK-3β signaling pathway, preventing wound healing, restraining cells migration from the upper chamber to the lower chamber. This study demonstrated that API can be used as a promising dietary supplement and an adjuvant chemotherapeutic agent for cancer treatment.

Juglone, a novel activator of ferroptosis, induces cell death in endometrial carcinoma Ishikawa cells.

Ferroptosis is a novel iron-dependent cell death pathway mainly caused by an abnormal redox state and associated with various diseases including cancer. Recently, much attention has been paid to natural compounds that are involved in its activation and inhibition. This is the first ever study to demonstrate the role of juglone isolated from Carya cathayensis green peel in inducing autophagy and inhibiting endometrial cancer (EC) cell migration. Subsequently, Fe2+ accumulation, lipid peroxidation, GSH depletion, the upregulation of HMOX1, and heme degradation to Fe2+ were reported. Juglone was involved in inducing autophagy and inhibiting cell migration and endoplasmic reticulum stress, which are the new hallmarks of cancer treatment. Collectively, our data indicate that juglone as a functional food ingredient induces the programmed cell death of EC cells by activating oxidative stress and suggest a novel therapeutic approach for the treatment and prevention of EC.

Plumbagin induces Ishikawa cell cycle arrest, autophagy, and apoptosis via the PI3K/Akt signaling pathway in endometrial cancer

Plumbagin (PLB) is a naphthoquinone endowed with potential medicinal properties, including anticancer activities. We evaluated the effects of PLB on the viability, cell cycle, autophagy, and apoptosis of endometrial carcinoma Ishikawa cells. The proliferation of cells was significantly inhibited by PLB at 0, 8, 10, and 12 μM. By up regulating the expression of p53 and p21, PLB could block the cell cycle in G2/M phase and down regulate cyclin dependent kinase. The apoptosis in the cancer cells was characterized by noticeable chromatin edge collection, nuclear membrane expansion, and vacuolization. PLB could significantly induce autophagy in cells, and its inhibition ability and apoptosis induction were weakened by the autophagy inhibitor SBI-0206965. Our study suggested that PLB may exert anticancer effects by abrogating PI3K/Akt pathway, which recommends it as a promising future phytotherapeutic candidate for EC treatment.

6-Shogaol mediated ROS production and apoptosis via endoplasmic reticulum and mitochondrial pathways in human endometrial carcinoma Ishikawa cells

Abstract Endometrial cancer (EC) associated with endogenous hormone metabolism, obesity, diabetes, and inappropriate dietary habits has shown a rising trend in recent years. Various therapeutics methods including dietary approaches (vital phytochemicals) offer healthy and nontoxic strategies against cancer. To date, 6-shogaol as one of the antitumor phytochemicals remains unexplored against human endometrial cancer cells. In our study, 6-shogaol obtained from Zingiber officinale rhizomes separated and purified by HPLC, LC-MS/MS, and NMR respectively could inhibit Ishikawa cell proliferation at IC50 (24.91 μM) with the arrested cell cycle in the G2/M phase. Moreover, it promoted apoptosis, as a result, triggered ROS production, then activated key ER response biomarkers in Ishikawa cell associated with mitochondria, and ultimately regulated related genes and proteins in vitro and in vivo. Therefore, 6-shogaol can be used as a promising natural compound endowed with potential benefits for cancer treatment and/or prevention at large and EC in particular.

2Fibulin-5 Inhibits Proliferation of Endometrial Carcinoma Ishikawa Cells by Down-Regulating β-Catenin

Endometrial cancer is a malignance in the uterus. The incidence of endometrial cancer is increasing recent years. Fibulin-5 participates in tissue homeostasis. The exact role of Fibulin-5 in endometrial cancer, such as becoming a sensitive tumor marker for endometrial cancer for diagnosis and follow-up, remains to be determined. 30 cases of endometrial tissue cancer specimens and adjacent tissues were collected for analysis of the expression of Fibulin-5 by western blot. Endometrial carcinoma Ishikawa cells (ECICs) were cultured and the level of Fibulin-5 was regulated via transfection. β-catenin level was measured followed by analysis of cell viability by MTT assay or BrdU staining. Results showed that Fibulin-5 in endometrial carcinoma tissues was significantly downregulated compared to para cancer tissues. Overexpression of Fibulin-5 decreased cell viability of endometrial carcinoma Ishikawa cells (ECICs) whereas downregulation increased cell viability. Meanwhile, Fibulin-5 overexpression significantly downregulated β-catenin and β-catenin knockdown blocked the proliferation effect caused by Fibulin-5 on ECICs. In conclusion, Fibulin-5 could inhibit cancer cell proliferation possibly via β-catenin related pathway.

MicroRNA-145-5p over-expression suppresses proliferation, migration and invasion and promotes apoptosis of human endometrial cancer cells by targeting dual specific phosphatase 6

To investigate the role of microRNA-145-5p (miR-145-5p) in regulating the proliferation, migration, invasion and apoptosis of human endometrial carcinoma cells. Human endometrial carcinoma Ishikawa cells were transfected with miR-145-5p mimic, miR-145-5p inhibitor, or their negative controls via liposome (Lipo2000), and the changes in the expression of miR-145-5p was verified by real-time PCR. The effects of overexpression or inhibition of miR-145-5p on the proliferation, migration, invasion and apoptosis of the cells were evaluated using MTT assay, wound healing assay, Transwell assay or flow cytometry. Bioinformatic analysis was performed to predict the target genes of miR-145-5p. The mRNA and protein expression levels of the downstream target of miR-145-5p, namely dual specific phosphatase 6 (DUSP6), were detected using real-time PCR and Western blotting. Transfection of the cells with miR-145-5p mimic significantly suppressed the proliferation of Ishikawa cells, while transfection with miR-145-5p inhibitor obvious enhanced the proliferation of the cells (P < 0.05). Over-expression of miR-145-5p significantly suppressed the migration and invasion and promoted apoptosis of the cells, and inhibition of miR-145-5p caused the reverse changes (P < 0.05). Bioinformatic analysis showed that DUSP6 was the potential target gene of miR-145-5p. Over-expression of miR-145-5p significantly lowered while inhibition of miR-145-5p significantly enhanced the expression of DUSP6 protein (P < 0.05). Overexpression of miR-145-5p inhibits the proliferation, migration and invasion and promotes apoptosis of endometrial cancer cells possibly by negative regulation of DUSP6 expression.

Expression Profiles of the Phosphatase and Tensin Homolog (PTEN), CDH1, and CDH2 Genes, and the Cell Membrane Protein, CD133, in the Ishikawa Human Endometrial Adenocarcinoma Cell Line.

BackgroundThis study aimed to investigate the expression profile of the phosphatase and tensin homolog (PTEN) gene, the cadherin genes, CDH1 and CDH2, and the cell membrane glycoprotein, CD133, in the Ishikawa human endometrial adenocarcinoma cell line.Material/MethodsThe Ishikawa endometrial carcinoma cell groups included cells transfected with the pLVX-puro lentiviral expression vector (the Ishikawa-puro group) and cells transfected with the pLVX-puro-PTEN lentiviral expression vector (the Ishikawa-PTEN group). The mRNA expression of the cadherin genes, CDH1 and CDH2, was detected by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The expression levels of the transmembrane glycoprotein CD133, a cancer stem cell marker, was detected by flow cytometry.ResultsThe expression of CDH1 and CDH2 mRNA in the Ishikawa-PTEN cells was lower than in the control cells. CD133 expression was lower in the Ishikawa-PTEN cells compared with the control cells.ConclusionsThis in vitro study showed that in Ishikawa endometrial carcinoma cells, downregulation of PTEN was associated with the expression of the CDH1 and CDH2 genes and upregulated expression of the cell membrane glycoprotein, CD133, which are associated with epithelial-mesenchymal transition (EMT) in malignancy. These findings support the need for further studies to investigate the potential role of PTEN in invasion and metastasis in endometrial carcinoma.

Nodal induces apoptosis and inhibits proliferation in ovarian endometriosis-clear cell carcinoma lesions

BackgroundExpression of Nodal, a member of the TGF-β superfamily, is commonly absent in differentiated tissues, while its re-expression occurs in a variety of human malignancy. However, little is known about its involvement in ovarian tumorigenesis. Herein, we focused on the functional roles of Nodal in ovarian endometriosis-carcinoma lesions.MethodsRegulation and function of Nodal and its associated molecules, including Smad2, GSK-3β, and several cell kinetics-related molecules, were assessed using clinical samples consisting of 108 ovarian carcinomas and 33 endometriotic lesions, as well as ES-2 (ovarian clear cell carcinoma; OCCCa) and Ishikawa (endometrial carcinoma) cell lines.ResultsNodal expression was significantly higher in endometriosis and OCCCa lesions as compared to that of non-OCCCas, with positive correlations to phosphorylated forms of both Smad2 (pSmad2) and GSK-3β. When compared to endometriotic lesions, the expression of Nodal and pSmad2 was significantly decreased in OCCCa. Treatment of Ishikawa cells with TGF-β1 resulted in transcriptional upregulation of Nodal, along with increased pSmad2 expression, while inhibition of GSK-3β also induced an increase in Nodal expression at the posttranslational level. Both ES-2 and Ishikawa cells stably overexpressing Nodal had increased susceptibility to apoptosis in response to treatment with cisplatin and doxorubicin, respectively, together with higher cleaved caspase-3 expression and decreased Bcl2/Bax ratio. Moreover, the stable Nodal-overexpressing cells showed reduced cell proliferation, along with increased expression of p27kip1 and p21waf1. In clinical samples, a significantly higher number of apoptotic cells and lower Ki-67 labeling indices were observed in Nodal-positive as compared to Nodal-negative OCCCa.ConclusionsThese findings suggest that Nodal is a multifunctional cytokine involved in the modulation of cell kinetics in ovarian endometriosis-OCCCa lesions.

The antitumor effect of lobaplatin against Ishikawa endometrial cancer cells in vitro and in vivo

The new effective chemotherapeutic drugs are required urgently for advanced and recurrent endometrial carcinoma (EC), which is one of the most common gynaecological tumors among women worldwide. Preclinical studies have shown that lobaplatin, one of the third-generation platinum compounds, has possessed powerful anti-cancer efficacy on a series of tumors. The purpose of this study is to investigate its effect and molecular mechanism on the growth of endometrial cancer cell line Ishikawa in vitro and in vivo. The results of cell counting kit-8 (CCK-8) shown that lobaplatin concentration-dependent inhibited cell proliferations in human endometrial carcinoma ishikawa cells. Flow cytometry (FCM) assay demonstrated that lobaplatin affected the survival of endometrial carcinoma cell by arresting cell cycle at S phase and G2/M phase and inducing apoptosis in dose-dependent manner. Moreover, Western blot analysis also showed that the apoptosis-inducing effects of lobaplatin was associated with the reduction of Bcl-2 expression while upregulation of cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9 and Bax. Meanwhile, lobaplatin significantly suppressed tumor growth of human Ishikawa xenograft models and terminal deoxynucleotidyl trans-ferase dUTP nick end labeling confirmed the significant occurrence of lobaplatin-treated tumor tissues of apoptosis. Therefore, lobaplatin could be an effective chemotherapeutic agent for human endometrial carcinoma and warrants further clinical investigation.

LncRNA TDRG1 enhances tumorigenicity in endometrial carcinoma by binding and targeting VEGF-A protein

Endometrial carcinoma is one of the most frequently diagnosed cancers in females. Long non-coding RNAs (lncRNAs) have been associated with cancer; its role in endometrial carcinoma is an emerging area of research. In this article, lncRNA TDRG1 expression in human endometrial carcinoma tissues and normal endometrial tissues was quantified by qRT-PCR. LncRNA TDRG1 was overexpressed or knocked-down in neither HEC-1B nor Ishikawa endometrial carcinoma cells, respectively, to assess cellular phenotype and expression of related molecules. Our results showed that lncRNA TDRG1 was significantly overexpressed in endometrial carcinoma tissues. Overexpression of lncRNA TDRG1 promoted endometrial carcinoma cell viability, invasion and migratory ability, inhibited apoptosis, and upregulated VEGF-A, PI3K, Bcl-2, MMP2 and survivin; knockdown of lncRNA TDRG1 had the opposite effects. LncRNA TDRG1 overexpression increased tumorigenicity in vivo and was associated with the upregulation of VEGF-A. RNA binding protein immunoprecipitation (RIP) assays confirmed that lncRNA TDRG1 directly binds to VEGF-A protein. Furthermore, knockdown of VEGFA in lncRNA TDRG1-overexpressing endometrial carcinoma cells reversed the effects of lncRNA TDRG1 on cell proliferation, invasion, migration and apoptosis. In conclusion, lncRNA TDRG1 may promote endometrial carcinoma cell proliferation and invasion by positively targeting VEGF-A and modulating relative genes.

Small interfering RNA-mediated knockdown of Twist attenuates the aggressive phenotypes of human endometrial carcinoma Ishikawa cells.

The present study aimed to determine the effect of Twist downregulation on the proliferation, apoptosis and migration of human endometrial carcinoma Ishikawa cells. Endogenous expression of the Twist transcription factor was knocked down by delivery of Twist-targeting small interfering RNA (siRNA). Changes in the expression of epithelial-mesenchymal transition biomarkers, namely epithelial (E)-cadherin, neural (N)-cadherin and Twist, were determined by western blot analysis. Cell cycle distribution and apoptosis were evaluated by flow cytometry. Cell proliferation and migration were analyzed using cell-counting and wound-healing assays, respectively. Transfection with Twist siRNA led to a significant reduction in the expression of Twist and N-cadherin (P<0.05), while significantly increasing the expression of E-cadherin, relative to negative control transfectants (all P<0.05). Proliferation was also significantly decreased in Ishikawa cells transfected with Twist siRNA (P<0.05), which was accompanied by an increased rate of apoptosis and cell cycle arrest at S-phase. In addition, Twist downregulation led to a significant reduction in cell migration (P<0.05). These data suggest that Twist serves a role in the regulation of cell proliferation and migration in Ishikawa cells and may represent a potential target for the treatment of human endometrial carcinoma.

Effects of 1alpha, 25-dihydroxyvitamin D3 on programmed cell death of Ishikawa endometrial cancer cells through ezrin phosphorylation

This study investigated the effects of 1α, 25-dihydroxyvitamin D3-induced cell death and its underlying molecular mechanisms in Ishikawa endometrial carcinoma cells. The effects of 1α, 25-dihydroxyvitamin D3 on Ishikawa cells were examined by 3-[4,5-dimethylthiazol-2-yl]-2.5-diphenyl-tetrazolium bromide, thiazolyl blue (MTT) assay. 1α, 25-dihydroxyvitamin D3 was shown to induce programmed cell death in Ishikawa endometrial carcinoma cells by activation of caspase-3 and caspase-9, along with elevation of Bcl-2 and Bcl-xL. Cell viability was reduced by 1α, 25-dihydroxyvitamin D3 in a concentration-dependent manner up to 2.5 μM. In addition, ezrin phosphorylation increased with the 1α, 25-dihydroxyvitamin D3 concentration (0–0.5 μM). The protein level of caspase-9 was increased by 1α, 25-dihydroxyvitamin D3 up to 0.5 μM. This is the first report regarding the efficacy and molecular mechanisms underlying the effects of 1α, 25-dihydroxyvitamin D3 in endometrial cancer cells. Our findings indicate that 1α, 25-dihydroxyvitamin D3 induces endometrial cancer cell death in a concentration-dependent manner.Impact statementUp to date, there is no report about the efficacy and molecular underlying mechanisms on the effect of vitamin D3 in endometrial cancer cells. Our findings indicate that 1α, 25-dihydroxyvitamin D3. which is an active metabolite of vitamin D3, induces Ishikawa endometrial cancer cell death in a concentration-dependent manner by activation of caspase-3 and -9, along with elevation of Bcl-2 and Bcl-xL. In addition, the same concentration of 1α, 25-dihydroxyvitamin D3 that provoked apoptotic signals caused phosphorylation of ezrin at threonine 567 in a VDR-dependent manner. This study suggests that 1α, 25-dihydroxyvitamin D3 within the optimal range (0.5 uM) would induce apoptosis through Fas-ezrin-caspase-3, -8, -9 signalling axis which may be a critical cell death regulator in Ishikawa endometrial cancer cell. Further study will be more interesting to address molecular connections or prove this critical optimal concentration range of vitamin D.

Fibulin-4 is associated with prognosis of endometrial cancer patients and inhibits cancer cell invasion and metastasis via Wnt/β-catenin signaling pathway.

Fibulin-4, an extracellular glycoprotein, which plays significant roles in elastic fiber assembly, is correlated to the progression of some cancers. However, the role of fibulin-4 in endometrial cancer cell invasion and metastasis remains unexplored. In our study, fibulin-4 expression was assessed by immunohistochemistry (IHC) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in normal endometrial tissues and endometrial carcinoma tissues. Using single cell cloning, strongly, and weakly, invasive subclones were derived from KLE and Ishikawa endometrial carcinoma cell lines. RT-qPCR, western blotting, and immunocytochemistry (ICC) were used to assess mRNA and protein expressions of fibulin-4 in primary cultured endometrial cells, 4 types of endometrial cancer cell lines, and the different invasive subclones. Using lentivirus transfection, fibulin-4 shRNA and pLVX-fibulin-4 were constructed and used to infect the strongly and weakly invasive subclones. The effects of fibulin-4 on the biological characteristics of endometrial carcinoma cells were detected by cell functional assays in vitro and in vivo. Using Wnt signaling pathway inhibitor XAV-939 and activator LiCl, we detected the role of fibulin-4 in the Wnt/β-catenin pathway and the relationship with epithelial to mesenchymal transition (EMT). Fibulin-4 was decreased in endometrial carcinoma tissues, and loss of fibulin-4 expression was significantly related with poor differentiation, lymph node metastasis, and poor prognosis of endometrial carcinoma. Fibulin-4 significantly inhibited endometrial carcinoma cell proliferation, invasion, metastasis, and EMT through the Wnt/β-catenin pathway. Fibulin-4 has the ability to suppress endometrial cancer progression. These results can contribute to the development of a new potential therapeutic target for patients with endometrial carcinoma.

Carfilzomib induces G2/M cell cycle arrest in human endometrial cancer cells via upregulation of p21Waf1/Cip1 and p27Kip1

ObjectiveCarfilzomib is a second-generation tetrapeptide epoxyketone proteasome inhibitor used in current clinical therapy of hematologic malignancies. The mechanism of proteasome inhibition in endometrial cancer is not very clear. Carfilzomib inhibition of type I endometrial carcinoma cell proliferation by inducing cell cycle arrest at the G2/M phase was investigated in our study. Materials and methodsHEC-1-A and Ishikawa endometrial carcinoma cell lines and three tumor cell lines were treated by different concentrations of carfilzomib. Methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability. Flow cytometry was used to analyze the cell cycle. Western blot was used to detect proteins involved in cell cycle progression. ResultsCarfilzomib impaired viability of myelogenous leukemia cell line K562, cervical cancer cell line HeLa, hepatocellular carcinoma cell line SMCC-7721, and endometrial carcinoma cell lines HEC-1-A and Ishikawa. The cell cycle was arrested at the G2/M phase in carfilzomib-treated HEC-1-A endometrial carcinoma cells, while it was arrested at both S and G2/M phases in carfilzomib-treated Ishikawa cells. Carfilzomib treatment significantly induced p21Waf1/ Cip1 and p27, while substantially reduced cyclin D3 and cyclin-dependent kinase 1. ConclusionThis study showed that carfilzomib inhibited endometrial cancer proliferation by upregulating cyclin-dependent kinase inhibitors p21Waf1/Cip1 and p27Kip1, and reducing cyclin-dependent kinase 1 to arrest the cell cycle at the G2/M phase.

Kruppel-like factor 4 (KLF-4) inhibits the epithelial-to-mesenchymal transition and proliferation of human endometrial carcinoma cells

Kruppel-like factors (KLFs) are a group of transcriptional regulators, being tumor-suppressive in various types of cancers, but not clear in human endometrial carcinoma (EC). We investigated the KLF-4 expression in both mRNA and protein levels in 29 EC specimens with RT-qPCR and Western blotting methods, and then to determine its promotion to Epithelial-to-mesenchymal transition (EMT) and proliferation of EC Ishikawa cells, via analyzing EMT-associated markers and via CCK-8 and colony forming assay. We found the downregulation of KLF-4 in the 29 EC specimens, correlating with the EC malignance. Moreover, we confirmed reduced levels of EMT and cell proliferation of Ishikawa cells post-KLF-4 overexpression. In conclusion, the significantly reduced KLF-4 correlated with the EC malignance. And the overexpressed KLF-4 promoted the EMT and proliferation of EC cells in vitro. The present study recognized the tumor suppressive role of KLF-4 in EC.