Esophageal Carcinoma Cell Lines Research Articles

The Level of Mesenchymal-Epithelial Transition Autophosphorylation is Correlated with Esophageal Squamous Cell Carcinoma Migration

Background:The MET receptor is a critical member of cancer-associated RTKs and plays an important role in different biological activities, including differentiation, migration, and cell proliferation. Methods:In this study, novel MET inhibitors were introduced and applied on esophageal squamous carcinoma cell line KYSE-30, and the level of proliferation and migration, as well as the activated form of MET receptor protein were assessed in the examined cells. The human KYSE-30 cell line was cultured according to ATCC recommendations. The mRNA level of the MET gene was measured in the examined cell line using the quantitative RT-PCR assay. Cytotoxicity evaluation test was performed at different concentrations of heterocyclic anti-MET compounds (i.e. D1, D2, D5, D6, D7, and D8). Finally, the capability of these compounds in MET receptor inhibition was evaluated using the migration assay and Western blot. All experiments were performed in triplicate and repeated three times with similar results. Results:Cell growth and proliferation were significantly inhibited (p ≤ 0.05) by all the above-mentioned compounds. Moreover, the majority of compounds significantly prevented the cell migration (p ≤ 0.05) and inhibited MET autophosphorylation. Interestingly, the level of phosphorylated MET was significantly correlated with KYSE-30 cell migration. Conclusion:The obtained data introduced and confirmed the biological activities of the mentioned novel compounds in KYSE-30 cells and proposed that the therapeutic inhibition of MET with these compounds may be a powerful approach for inhibiting cancer cell migration and proliferation although some structural optimizations are needed to improve their inhibitory functions.

Circular RNA 0014715 Facilitates Cell Proliferation and Inhibits Apoptosis in Esophageal Squamous Cell Carcinoma.

BackgroundCircular RNAs (circRNAs) have recently been verified to have multiple biological functions and participate in diverse biological processes in different malignant tumors, including esophageal squamous cell carcinoma (ESCC). Nonetheless, the function of circular RNA 0014715 (hsa_circ_0014715, circ_0014715) in ESCC has not been described.Materials and MethodsWe investigated clinical data from sixty-seven patients undergoing surgery for esophageal cancer. The clinical data were collected. And we analyzed the correlation between the clinical characteristics of these patients and the expression of circ_0014715. Besides, we explored the expression of circ_0014715 in ESCC cell lines. We used cell counting kit-8, colony formation, transwell assay, and flow cytometry to detect changes in cell proliferation, migration, apoptosis, and cell cycle progression.ResultsWe found that circ_0014715 was highly expressed in esophageal squamous cell carcinoma tissues and cell lines. The correlation analysis of clinicopathological features and gene expression revealed that high expression of circ_0014715 was related to nerve invasion, vascular invasion, more advanced tumor-node-metastasis (TNM) stage and poor differentiation grade. Receiver operating characteristic (ROC) curves revealed that circ_0014715 might have diagnostic value for ESCC. Experiments with cultured cells showed that knockdown of circ_0014715 significantly restrained cell proliferation, migration, invasion, wound healing and accelerated cell apoptosis. And cell cycle arrest at G2 phase was observed via flow cytometry. Overexpression of circ_0014715 caused the opposite effects. Collectively, these studies show that circ_0014715 is closely connected with the pathogenesis and development of ESCC. The excess expression of circ_0014715 may have promoting effects on the progression of esophageal cell carcinoma.ConclusionOur finding revealed that circ_0014715 promoted tumor growth and cell proliferation. All of these suggest that targeting circ_0014715 has potential therapeutic value in the treatment of ESCC.

Metastatic Esophageal Carcinoma Cells Exhibit Reduced Adhesion Strength and Enhanced Thermogenesis.

Despite continuous improvements in multimodal therapeutic strategies, esophageal carcinoma maintains a high mortality rate. Metastases are a major life-limiting component; however, very little is known about why some tumors have high metastatic potential and others not. In this study, we investigated thermogenic activity and adhesion strength of primary tumor cells and corresponding metastatic cell lines derived from two patients with metastatic adenocarcinoma of the esophagus. We hypothesized that the increased metastatic potential of the metastatic cell lines correlates with higher thermogenic activity and decreased adhesion strength. Our data show that patient-derived metastatic esophageal tumor cells have a higher thermogenic profile as well as a decreased adhesion strength compared to their corresponding primary tumor cells. Using two paired esophageal carcinoma cell lines of primary tumor and lymph nodes makes the data unique. Both higher specific thermogenesis profile and decreased adhesion strength are associated with a higher metastatic potential. They are in congruence with the clinical patient presentation. Understanding these functional, biophysical properties of patient derived esophageal carcinoma cell lines will enable us to gain further insight into the mechanisms of metastatic potential of primary tumors and metastases. Microcalorimetric evaluation will furthermore allow for rapid assessment of new treatment options for primary tumor and metastases aimed at decreasing the metastatic potential.

Knockdown of NOLC1 Inhibits PI3K-AKT Pathway to Improve the Poor Prognosis of Esophageal Carcinoma.

Objective Esophageal carcinoma (ESCA) is a common malignant gastrointestinal tumor. The abnormal expression of NOLC1 is involved in the tumorigenesis of various human tumors, whereas the function and mechanism of NOLC1 in ESCA remain unclear. In this study, we explored the relationship between NOLC1 and poor prognosis of ESCA, and its role and mechanism in the occurrence of ESCA. Methods The NOLC1 expression in ESCA tissues and cell lines was determined by qRT-PCR, immunohistochemistry, or western blot. The Kaplan–Meier method was conducted to estimate the overall survival. Cox regression analysis was carried out to examine the association between patient characteristics and prognosis. A recombined lentiviral vector containing NOLC1 was applied for transfecting ESCA cells (Eca109 and TE-13) and established a stable cell line with low NOLC1 expression or high NOLC1 expression, in the absence or presence of PI3K inhibitor (LY294002) treatment. Cell proliferation, apoptosis rate, invasion ability, migration ability, and PI3K/AKT pathway were detected by CCK8 assay, flow cytometry, Transwell assay, wound-healing assay, and western blot. Results NOLC1 overexpression was observed in ESCA tissues and ESCA cell lines (EC9706, Eca109, TE-13, Kyse170, T.TN) compared with adjacent normal tissues and normal esophageal cell line HEEC. NOLC1 overexpression was markedly associated with bigger tumor size, lymph node metastasis, and advanced TNM stage. Patients with NOLC1 overexpression have shorter overall survival than that of those with low NOLC1 expression. NOLC1 overexpression was considered to be an independent poor prognostic factor affecting overall survival. NOLC1 knockdown inhibited proliferation, migration, invasion, and cyclin B1 expression and promoted the apoptosis and cleaved-caspase-3 expression of Eca109 and TE-13 cells. NOLC1 overexpression accelerated proliferation, migration, invasion, and cyclin B1 expression and inhibited the apoptosis and cleaved-caspase-3 expression of ESCA cells via activating PI3K/AKT pathway. Rescue experiments showed that PI3K inhibitor (LY294002) could reverse the phenomenon caused by NOLC1 overexpression. Conclusion NOLC1 may be a marker for poor prognosis. It can participate in the occurrence and development of ESCA via the PI3K/AKT pathway.

Qigefang Inhibits Migration, Invasion, and Metastasis of ESCC by Inhibiting Gas6/Axl Signaling Pathway.

In recent years, there is an increasing interest in using Traditional Chinese Medicine (TCM) and their patents for the treatment of cancers. Qigefang (QGF) is a TCM formula and has been used for the treatment of metastatic esophageal cancer in China. However, its therapeutic effect on tumors and its mechanism of action is largely unknown. The aim of this study is to explore the role of QGF in the treatment of metastasis of Esophageal Squamous Cell Carcinoma( ESCC). Human esophageal carcinoma cell line KYSE150 was used for this study. CCK-8 assay was used to determine the cytotoxicity of QGF. The KYSE150 cells were treated with QGF to determine its effect on cell migration (cell scratch assay and imaging) and invasion (Transwell system based with Matrigel assay). Western blotting was used to investigate the effect of QGF on relevant molecules of signaling pathways. A mouse model of lung metastasis of esophageal cancer was established by injecting the KYSE150-Luc cells through the tail vein. A small animal imaging system was used to observe tumor metastasis in the mice. QGF reduced cell migration and invasion of KYSE150 cells. QGF significantly inhibited lung metastasis in nude mice. Further study revealed that the expression of Growth arrest-specific 6 (Gas6), Anexelekto (Axl), N-Nuclear factor-kappa B (NF-κB) and matrix metalloproteinase-9 (MMP-9) proteins were decreased both in vitro and in vivo upon treatment with QGF. QGF could prevent invasion and metastasis of esophageal cancer by inhibiting the Gas6/Axl signaling pathway.

Mitochondrial pathway of the lysine demethylase 5C inhibitor CPI-455 in the Eca-109 esophageal squamous cell carcinoma cell line.

BACKGROUNDEsophageal cancer is a malignant tumor of the digestive tract that is difficult to diagnose early. CPI-455 has been reported to inhibit various cancers, but its role in esophageal squamous cell carcinoma (ESCC) is unknown.AIMTo investigate the effects and mechanism of the lysine demethylase 5C inhibitor, CPI-455, on ESCC cells.METHODSA methyl tetrazolium assay was used to detect the inhibitory effect of CPI-455 on the proliferation of Eca-109 cells. Apoptosis, reactive oxygen species (ROS), and mitochondrial membrane potential were assessed by flow cytometry. Laser confocal scanning and transmission electron microscopy were used to observe changes in Eca-109 cell morphology. The protein expression of P53, Bax, lysine-specific demethylase 5C (KDM5C), cleaved Caspase-9, and cleaved Caspase-3 were assayed by western blotting.RESULTSCompared with the control group, CPI-455 significantly inhibited Eca-109 cell proliferation. Gemcitabine inhibited Eca-109 cell proliferation in a concentration- and time-dependent manner. CPI-455 caused extensive alteration of the mitochondria, which appeared to have become atrophied. The cell membrane was weakly stained and the cytoplasmic structures were indistinct and disorganized, with serious cavitation when viewed by transmission electron microscopy. The flow cytometry and western blot results showed that, compared with the control group, the mitochondrial membrane potential was decreased and depolarized in Eca-109 cells treated with CPI-455. CPI-455 significantly upregulated the ROS content, P53, Bax, Caspase-9, and Caspase-3 protein expression in Eca-109 cells, whereas KDM5C expression was downregulated.CONCLUSIONCPI-455 inhibited Eca-109 cell proliferation via mitochondrial apoptosis by regulating the expression of related genes.

TRPC1 Inhibits Cell Proliferation/Invasion and Is Predictive of a Better Prognosis of Esophageal Squamous Cell Carcinoma.

Background and ObjectivesIn China, over 90% of esophageal cancer (EC) cases are esophageal squamous cell carcinoma (ESCC). ESCC is a frequently malignant tumor with poor prognosis despite the development of comprehensive therapeutic strategies, for which there is still a lack of effective prognostic factors. Previous studies found that the abnormal expression of TRPC1 is closely related to the proliferation, invasion, metastasis, and differentiation of various tumors. However, the relationship between TRPC1 and ESCC is currently unclear. The present study aimed to clarify the clinical significance of TRPC1 and to preliminarily assess the molecular mechanism by which TRPC1 regulates cell proliferation, migration, and invasion in ESCC.Materials and MethodsImmunohistochemistry (IHC) was used to determine the expression of TRPC1 and Ki-67 in 165 cases of ESCC. The correlations between TRPC1 expression and clinicopathological characteristics were determined, and both univariate and multivariate analyses were utilized to quantify the impact of TRPC1 expression on patient survival. Cell Counting Kit-8, scratch wound healing, and transwell assays were used to determine the effects of TRPC1 on proliferation, migration, and invasion in ESCC in vitro, respectively.ResultsThe positive expression rate of TRPC1 showed significantly decreased in ESCC (45.50%) compared with the levels in normal esophageal mucosa (NEM; 80.80%) and high-grade intraepithelial neoplasia (HGIEN; 63.20%) (P<0.001). Higher expression rate of TRPC1 was associated with low lymph node metastasis (P<0.001), high differentiation (rs = 0.232, P=0.003), and low Ki-67 (rs = −0.492, P<0.001). We further revealed that low expression of TRPC1 was associated with poor prognosis (Disease-free survival, DFS: 95% CI=0.545–0.845, P=0.001; Overall survival, OS: 95% CI=0.553–0.891, P=0.004). Furthermore, we showed that downregulation of TRPC1 promoted the proliferation, migration, and invasion of human esophageal squamous cell carcinoma cell line EC9706 in vitro. In contrast, overexpression of TRPC1 inhibited the proliferation, migration, and invasion of human esophageal squamous cell carcinoma cell line KYSE150 (P<0.01), in a manner at least in part mediated through the AKT/p27 pathway.ConclusionTRPC1 inhibited the proliferation, migration, and invasion of EC9706 and KYSE150 cells, at least, in part mediated through the AKT/p27 pathway in vitro. The downregulation of TRPC1 may be one of the most important molecular events in the malignant progression of ESCC. TRPC1 could be a new candidate tumor suppressor gene and a new prognostic factor of ESCC.

BAP1 promotes viability and migration of ECA109 cells through KLF5/CyclinD1/FGF-BP1.

More than 40 000 patients worldwide die from esophageal cancer annually. The 5‐year survival rate of patients is only ~ 15–20%, and thus, there is an ongoing need to improve diagnosis and treatment of esophageal cancer. Breast cancer type 1 susceptibility protein (BRCA1)‐associated protein (BAP1) is a marker of poor prognosis in several cancers, including uveal melanoma, renal cell carcinoma, cholangiocarcinoma, non‐small cell lung cancer, and colorectal cancer. BAP1 mutations are early and rare events in esophageal carcinoma, but the involvement of BAP1 in progression of esophageal carcinoma is unclear. Here, we report that cell proliferation and migration were significantly enhanced in esophageal carcinoma ECA109 cells overexpressing BAP1, while they were diminished upon BAP1 knockdown. In addition, the expression of Krüppel‐like factor 5 (KLF5), CyclinD1, and FGF‐BP1 was increased by BAP1 overexpression and decreased by BAP1 knockdown. Our data suggest that BAP1 promotes cell proliferation and migration, and enhances the expression of KLF5 and its downstream genes, including CyclinD1 and FGF‐BP1, in the esophageal carcinoma cell line ECA109.

Green synthesis of NiO nanoparticles using Calendula officinalis extract: Chemical charactrization, antioxidant, cytotoxicity, and anti-esophageal carcinoma properties

In this study, nickel nanoparticles (NiONPs) have been successfully synthesized in an aqueous medium using the aqueous extract from the leaves of Calendula officinalis. The synthesized NiONPs were characterized using different techniques. According to the XRD analysis, the mean crystal size for NiONPs was found to be 33.17 nm. In addition, SEM images exhibited a uniform spherical morphology with a size of 60.39 nm for the NiONPs. The anti-esophageal carcinoma potentials of C. officinalis leaf aqueous extract, and NiONPs were assessed by the MTT assay against distal esophageal adenocarcinoma (FLO-1), gastroesophageal junction adenocarcinoma (ESO26), human caucasian esophageal carcinoma (OE33), and human esophageal squamous cell carcinoma (KYSE-270) cell lines. This study showed that NiONPs had excellent cell death and anti-cancer effects against esophageal carcinoma cell lines. The IC50 of NiONPs were 380, 263, 229, and 251 µg/mL against FLO-1, ESO26, OE33, and KYSE-270 cell lines, respectively. Among the studied cell lines, the best anti-esophageal cancer activities of NiONPs were gained in the cell line of OE33. The antioxidant activity of NiONPs was investigated using the DPPH test. In the antioxidant test, NiONPs prevented the oxidation of 50% of the DPPH molecules at a concentration of 204 µg/mL. Considering the obtained results, it can be suggested that NiONPs may be administrated as a chemotherapeutic supplement/drug to treat esophageal carcinoma.

Docetaxel Toxicity Optimization in Esophageal Squamous Cell Carcinoma Cell Line YM-1: A Study of Cell Cycle and Doubling Time Effect

Docetaxel Toxicity Optimization in Esophageal Squamous Cell Carcinoma Cell Line YM-1: A Study of Cell Cycle and Doubling Time Effect

Beta-Elemene Suppresses Metastasis of Human Esophageal Cancer Cell by Downregulating Heat Shock Protein 70/Mothers against Decapentaplegic Homolog 2 Pathway

In this study, we tried to illuminate the role and underlying mechanism of beta-elemene in esophageal squamous cell carcinoma especially the metastasis. 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide assay was performed to determine the cytotoxicity of beta-elemene and its role in esophageal squamous cell carcinoma cell proliferation. Wound healing assay and transwell assays were used to elucidate the roles of betaelemene on cell migration and invasion abilities of esophageal squamous cell carcinoma cells. Quantitative polymerase chain reaction and western blot assays were carried out to assess the messenger RNA and protein expression under beta-elemene treatment. Immunofluorescence staining was performed to detect the heat shock protein 70 expression regulated by beta-elemene. The heat shock protein 70 ectopic expression plasmid was used to perform the rescue experiments. In this study, we found that beta-elemene significantly inhibited the cell survival and cell proliferation and suppressed cell migration and invasion abilities of esophageal squamous cell carcinoma cell line. Mechanistically, we observed that beta-elemene downregulated heat shock protein 70 expressions and reversed epithelial-mesenchymal transition. Furthermore, the studies also indicated that beta-elemene downregulated the phosphorylation of extracellular regulated kinase, mothers against decapentaplegic homolog 2 and mothers against decapentaplegic homolog 3 expression. In addition, we confirmed that ectopic expression of heat shock protein 70 not only promoted cell migration, epithelialmesenchymal transition and mothers against decapentaplegic homolog 2 phosphorylation in esophageal squamous cell carcinoma cell line cells, but also reversed beta-elemene suppressed cell migration in TE-1 cells. Taken together, all results elucidated that beta-elemene suppresses metastasis of human esophageal squamous cell carcinoma by modulating the expression of heat shock protein 70 and the activation of extracellular regulated kinase and transforming growth factor beta signaling pathways at least partially.

MiR‑93‑5p regulates the occurrence and development of esophageal carcinoma epithelial cells by targeting TGFβR2.

Emerging studies have indicated that the dysregulation of microRNAs (miRNAs or miRs) plays a vital role in the development and metastasis of tumors. However, the role of miR-93-5p in esophageal carcinoma (EC) has not been extensively reported. The present study thus focused on the role of miR-93-5p and its downstream target in the occurrence and development of EC. Firstly, miRNA expression profiles associated with EC were accessed from the TCGA_ESCA dataset and analyzed. Subsequently, the expression patterns of miR-93-5p and TGFβR2 were characterized in the human esophageal cell line, Het-1A, and the human EC cell lines, TE-1, Eca-109 and EC9706, by RT-qPCR and western blot analysis. WST-1 assay, flow cytometry, Transwell assay, wound healing assay and bioinformatics analysis were used to explore their functions in EC cells. Finally, a dual-luciferase reporter assay was employed to determine the targeted association between miR-93-5p and TGFβR2. The results revealed that the expression of miR-93-5p was markedly higher in EC cell lines compared with that in the normal cell line. The overexpression of miR-93-5p facilitated cell proliferation, migration and invasion, and inhibited cell apoptosis. Additionally, TGFβR2 was identified as a functional target of miR-93-5p in EC cells, as judged by a series of in vitro experiments. Furthermore, it was found that the simultaneous overexpression of miR-93-5p and TGFβR2 almost had no effect on the biological behaviors of EC cells. On the whole, the present study demonstrates that miR-93-5p promotes the proliferation, migration and invasion, and inhibits the apoptosis of EC cells by targeting TGFβR2.

High expression of collagen 1A2 promotes the proliferation and metastasis of esophageal cancer cells.

BackgroundTo undertake a bioinformatics analysis to identify abnormally expressed genes [also referred to as differentially expressed genes (DEGs)] and their functions in esophageal carcinoma (ESCA).MethodsDEGs (i.e., GSE100942, GSE17351, GSE26886, and GSE77861) were obtained from a gene expression omnibus database. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using online tools from the Database for Annotation, Visualization and Integrated Discovery. A protein-protein interaction network was then constructed based on the Search Tool for the Retrieval of Interacting Genes website. Cytoscape software was used to identify the top 20 DEGs located in the central region of the network. For the overall survival analysis, a Kaplan–Meier analysis was conducted of the Gene Expression Profiling Interactive Analysis website, and collagen (COL) 1A2 was selected to detect the molecular mechanism of COL1A2-small interfering ribonucleic acid (siRNA) in the following ESCA cell lines: Eca109 and TE-1. Next, the expression of COL1A2-messanger ribonucleic acid was determined using real-time quantitative polymerase chain reaction. The expression of COL1A2 was also verified by Western blot. Cell proliferation was measured by colony-forming and MTT assays, and migration and invasion by the transwell assay.ResultsBased on the GEO database and screening out the hub gene, we identified that COL1A2 was abnormally expressed in ESCA. With a series of in vitro experiments, the expression of COL1A2 was defined as higher in Eca109 and TE-1.ConclusionsCOL1A2 was highly expressed in ESCA tissue samples. Additionally, the proliferation and metastasis of Eca109 and TE-1 cell lines were significantly attenuated by siRNA-COL1A2-mediated small interference. Notably, the expression level of COL1A2 was obviously related to the Akt and epithelial-mesenchymal transition (EMT) pathways.

Effect of miR-146a on biological behavior of esophageal squamous cell carcinoma cells and its mechanism

Objective: To investigate the effects and mechanisms of miR-146a on the proliferation, invasion, migration, apoptosis and cell cycle of esophageal squamous cell carcinoma cells. Methods: The expressions of miR-146a in 3 esophageal squamous carcinoma cell lines (Eca109, KYSE140, KYSE150) were detected by real-time quantitative polymerase chain reaction (RT-qPCR). MiR-146a mimics was transfected into Eca109 to up-regulate the expression of miR-146a. Effects of miR-146a on cell proliferation, invasion and migration were evaluated by cell counting kit 8 (CCK-8), Transwell assay and wound-healing assay, respectively. The cell apoptosis and cycle were assessed by flow cytometry (FCM). Finally, relevant bioinformatics techniques were used to predict the target gene of miR-146a. Dual luciferase reporter assay was used to identify the interaction of 3' terminal untranslated region (3' UTR) of miR-146a and its target gene, interleukin-1 receptor-associated kinase (IRAK1). RT-qPCR and western blot were used to detect the mRNA and protein expressions of IRAK1, respectively. Results: The relative expressions of miR-146a in 3 esophageal squamous cell carcinoma cells (Eca109, KYSE140, KYSE150) were 0.36±0.05, 0.16±0.06 and 0.09±0.02, respectively, all of which were significantly lower than 1±0.05 of normal esophageal epithelial cells (HEEC) (P<0.01). The model of esophageal squamous cell carcinoma cells was constructed by transfecting miR-146a mimics into Eca109 cells. The results showed that the ability of absorbance value, the number of transmembrane cells (52±18), the reduced scratch distances at 48 hours and 72 hours after transfection [(25.29±0.77) μm, (30.66±0.91) μm] were significantly lower than those of the negative control group and blank control group (all P<0.01). The early apoptosis rate was (6.13±0.91)%, higher than (2.50±0.68)% of the negative control group (P<0.01) and (1.70±0.20)% of blank control group (P<0.01). The percentage of cells in G(1) phase [(44.74±6.76)%] was decreased while the G(2)/M phase [(41.88±2.88)%] was increased when compared with the negative control group and the blank control group (P<0.05 and P<0.01, respectively). The results of dual luciferase reporter gene assay showed that luciferase activity in the group co-transfected with IRAK1-wild-type and miR-146a mimics was significantly lower than that in the control groups (P<0.01). The results of RT-qPCR and western blot showed that the mRNA and protein expressions of IRAK1 in the co-transfected group were 1.02±0.28 and 1.00±0.05, respectively, both lower than those in the negative control group and the blank control group (P<0.01). Conclusions: The expressions of miR-146a are decreased in the esophageal squamous cell lines, which plays a role as tumor suppressor gene. MiR-146a can inhibit the proliferation, invasion and migration of esophageal squamous cell cells, promote apoptosis, and block the cell cycle at G(2)/M stage. MiR-146a may mediate the malignant biological behavior of esophageal cancer cells through the regulation of IRAK1.

Evaluation and Optimization of Lipofectamine 3000 Reagents for Transient Gene Expression in KYSE-30 Esophagus Cancer Cell Line

Background and Aim Transfection of DNA/RNA sequence into eukaryotic cells has a major effect on scientific studies. Various methods are used to transfer the DNA/RNA sequence into cells, such as lipid-based carriers as the available and easy procedure. Transfection with cationic lipid liposome is introduced as a simple and efficient procedure for monitoring the DNA/RNA sequence through gene function analysis, including fluorescence imaging RNA and protein expression. This study aimed to investigate the transfection efficiency and cell death through GFP expression in human esophageal squamous cell carcinoma (ESCC) cell line KYSE-30 using Lipofectamine 3000 reagent. Methods The pCDH-513b plasmid DNA was transfected into KYSE-30 cells using Lipofectamine 3000 in different concentrations of the plasmid DNA and reagent. The transfection efficiency was evaluated by fluorescence microscope and flow cytometry analysis to determine the percentage of GFP-expressing cells. Moreover, the viability and death of transfected KYSE-30 cells were evaluated using a trypan blue exclusion assay. Results The transfection efficiency of KYSE-30 with Lipofectamine 3000 was increased with higher plasmid DNA concentration and a lower amount of Lipofectamine 3000 reagent. The Optimized concentration of 1.5 µg plasmid DNA and volume of one µl of lipofectamine 3000 reagents were identified for 95% transfection efficiency in the KYSE-30 cell line. The viability and death of transfected cells were 43% and 58% after transfection, respectively. Conclusion The results indicated that Lipofectamine 3000 might not be suitable for transfection in KYSE-30 cells due to increased cell death.

The novel target of esophageal squamous cell carcinoma: lncRNA GASL1 regulates cell migration, invasion and cell cycle stagnation by inactivating the Wnt3a/β-catenin signaling

Long non-coding RNA (lncRNA) Growth-Arrest Associated LncRNA 1 (GASL1) is a lncRNA with a suppressive role in glioma, prostate carcinoma and gastric carcinoma, whereas its involvement in esophageal cancer is unknown. In the present study, we used RT-qPCR to detect the expression of GASL1 in esophageal cancer cell carcinoma (ESCC) cell lines, and constructed the overexpression and interference plasmids of GASL1 and the interference plasmid of DKK1. CCK8 was used to detect the cell proliferation level, clone formation experiment was used to detect the cell clonal formation ability, flow cytometry was used to detect the cell cycle level, and wound healing and Transwell experiments were respectively used to detect the cell invasion and migration. The interference and overexpression plasmids of GASL1 were injected into mice subcutaneously for tumor-bearing experiment. The body weight, tumor growth curve, and tumor weight of mice were recorded, and western blot was used to detect the expression of proliferation-, invasion-, and migration-related proteins and the expression of Wnt3a/β-catenin signal-related proteins in tumor tissues. LncRNA GASL1 was down-regulated in ESCC cell lines, and GASL1 inhibited ESCC cell progression and regulated cell cycle arrest in ESCC cells. In vivo, GASL1 inhibited tumor growth. GASL1 decreased the protein levels of DDK1, Wnt3a, β-catenin, and c-MYC in ESCC cell lines. Interfering DKK1 activates Wnt3a/β--catenin signal to reverse the inhibitory effects of GASL1 on proliferation, cell cycle acceleration, invasion, and migration. In conclusion, lncRNA GASL1 regulates cell migration, invasion and cell cycle stagnation by inactivating the wnt/β-catenin signaling.

Inhibitory Effect of Eicosapentaenoic Acid on the Migration of the Esophageal Squamous Cell Carcinoma Cell Line TE-1.

Eicosapentaenoic acid (EPA) inhibits NF-ĸB activation and IL-6 production in TE-1 esophageal cancer cells. NF-ĸB is related to cancer cell migration. The aim of this study is to evaluate whether EPA has a metastasis suppressing effect. Herein, we investigated EPA-treated TE-1 cell migration using TAXIScan. EZ-TAXIScan® was used to verify whether EPA inhibits cancer cell chemotaxis. Using 50% fetal bovine serum (chemoattractant) without EPA (positive control), average velocity was 0.306±0.084 μm/min compared to 0.162±0.067 μm/min without chemoattraction (negative control). Directionalities of positive and negative controls were 1.039±0.152 and 0.488±0.251 radians, respectively, indicating a significant increase in migration of the positive control compared to that of the negative control. Average velocities were 0.306±0.084 (no EPA), 0.288±0.078 (100 μM EPA), and 0.240±0.054 200 μM (EPA) μm/min, indicating that EPA reduced velocity dose-dependently. Average directionalities were 1.039±0.152 (no EPA), 0.967±0.164 (100 μM EPA), and 0.901±0.146 (200 μM EPA) radians, indicating that EPA also inhibited directionality dose-dependently. EPA suppresses directional migration of TE-1 cells.

CDKL3 Targets ATG5 to Promote Carcinogenesis of Esophageal Squamous Cell Carcinoma.

ObjectiveOur previous study suggested cyclin-dependent kinase-like 3 (CDKL3) acts as a new oncogene in esophageal squamous cell carcinoma (ESCC) cell line TE-1. However, the molecular mechanisms and biological effects of CDKL3 in ESCC remain unknown. In the present study, we aimed to explore the clinical significance of CDKL3 in ESCC and how CDKL3 regulates the malignant behavior of ESCC.MethodsESCC samples were stained by immunohistochemical staining (IHC) and analyzed for the expression of CDKL3. The functions of CDKL3 on proliferation, apoptosis, migration, invasion, and colony formation were investigated by celigo assay, MTT assay, colony formation, caspase 3/7 activity analysis, transwell migration and invasion assay, respectively. A transplanted tumor model was established to study the functions of CDKL3 on the tumorigenesis of ESCC cells. Microarray analysis was utilized to identify the CDKL3-regulated genes in ESCC cells.ResultsESCC tumor tissues possessed a significantly higher expression of CDKL3 and autophagy-related gene 5 (ATG5) than matched adjacent normal tissues. The high expressions of CDKL3 were positively associated with the tumor-node-metastasis (TNM) stage and Ki67. Upregulated ATG5 expression was positively correlated with male, advanced tumor (T) stage, and TNM stage. Kaplan-Meier analysis showed that ESCC patients with higher expression of CDKL3 or ATG5 had a shorter overall survival. The worst prognosis was recognized in patients with both high manifestations of CDKL3 and ATG5. Time-dependent receiver operating characteristic (ROC) curve was established to reveal that the combination of CDKL3, ATG5, and TNM stage–based model had better prognostic accuracy than TNM stage. Moreover, CDKL3 knockdown markedly repressed cell growth and aggressivity in vitro and in vivo. Mechanistically, ATG5 was confirmed as a downstream gene involved in the pro-oncogenic function of CDKL3.ConclusionCDKL3 can be utilized as an independent poor prognostic marker in ESCC patients. Furthermore, CDKL3 may promote tumor profession, invasion, metastasis, and prohibit tumor apoptosis partly by ATG5 activation.

Abstract 1854: Oridonin inhibits cell growth of esophageal carcinoma cells via induction of apoptosis

Abstract Esophageal Cancer estimated leading deaths are more than 400,000 every year in worldwide, while 80% due to esophageal squamous cell carcinoma. In worldwide, esophageal cancer leading death ranks in sixth among cancer leading causes of death, while the 5-year survival rate of esophageal cancer is 19%. Oridonin, a tetracyclic diterpenes compound, has been indicated the anti-tumor activity for digestive system tumors. To assess the anticancer activity of oridonin on esophageal cancer, we performed cell proliferation experiment of oridonin on esophageal squamous cell carcinoma cell line TE-8 using SRB assay. We found that IC50 of oridonin on TE-8 treated for 72h is 3.0μM, which is better than positive control Gemcitabine treatment on TE-8 for 72h (5.7 μM), as well as TE-8 cells shrunk and floated with increasing concentration on oridonin. At the same time, oridonin induced a dose-dependent inhibition of cell colony formation of esophageal cancer cell line. Colony numbers of TE-8 cell line treated with 1μM and 2μM oridonin are 111 and 54, respectively, which are significantly different (p&amp;lt;0.05). Accompanying with marked morphological changes, flow cytometer results showed that oridonin triggered sub-G0/G1 cell cycle arrest in a dose-dependent manner. After treatment by 10μM and 20μM oridonin for 24h, percentage of sub-G0/G1 cells were 25.5 and 55.3%, respectively, which are significantly different (p&amp;lt;0.05). The apoptosis results showed that oridonin treatment led to the accumulation of cells in early-(Annexin V+/PI-) and late-stage (Annexin V+/PI+) apoptosis in a dose-dependent manner. Citation Format: Xiaojun Zhang, Mengtao Xing, Yangcheng Ma, Xiao Wang, Jianying Zhang. Oridonin inhibits cell growth of esophageal carcinoma cells via induction of apoptosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1854.

Interaction of Estradiol and Endoplasmic Reticulum Stress in the Development of Esophageal Carcinoma.

Gender differences in esophageal cancer patients indicate that estradiol may have antitumor effects on esophageal cancer. The initiation of endoplasmic reticulum stress (ERS) can induce apoptosis in esophageal cancer cells. However, it is still unknown whether estradiol inhibits the development of esophageal cancer by activating ERS pathway. In this study, the gender difference in the development of esophageal cancer was observed by analyzing clinical data and the experimental tumor xenografts in mice. Meanwhile, we investigated the mechanism of ERS in estradiol-mediated inhibition of esophageal cancer using esophageal squamous cell carcinoma cell line EC109. The proportion of male patients with esophageal cancer was significantly higher than female patients. Meanwhile, male patients were prone to have adventitial invasion. The weight of transplanted tumors in female mice was significantly smaller than that in male mice. In vitro experiments showed estradiol inhibits the viability and migration of EC109 cells by increasing the expression of ERS-related proteins, whereas ERS inhibitor 4-PBA abolished the effects of estradiol. In conclusion, our data demonstrate that sex difference exists in the occurrence of esophageal cancer. Estradiol can inhibit the viability and migration of esophageal cancer cells through the activation of ERS, providing a novel insight for esophageal cancer development, treatment, and prevention.