Esophageal Carcinoma Eca-109 Cells Research Articles

The Diurnal Transcriptome Reveals the Reprogramming of Lung Adenocarcinoma Cells Under a Time-Restricted Feeding-Mimicking Regimen

BackgroundThe processes of tumor growth and circadian rhythm are intimately intertwined; thus, rewiring circadian metabolism by time-restricted feeding (TRF) may contribute to delaying carcinogenesis. However, research on the effect of a TRF cellular regimen on cancer is lacking. ObjectiveInvestigate the circadian signatures of TRF in lung cancer in vitro. MethodsWe first developed a cellular paradigm mimicking in vivo TRF and collected cells for transcriptome analysis. We further confirmed the effect on tumor cells upon 6-h TRF-mimicking (6-h TRFM) by real-time PCR, Lumicycle experiments, CCK-8, and flow cytometry assays. ResultsWe found that A549 lung adenocarcinoma cells treated with 6-h TRFM conditions displayed robust diurnal rhythms of transcriptomes, as well as modulation of the core clock genes relative to other different cellular regimens used in this study, including the fasting-mimicking conditions (ie, short-term starvation) and the serum-free regime. Notably, pathway analysis of oscillating genes exclusively in 6-h TRFM showed that some circadian genes were enriched in tumor-related pathways, such as the oxytocin signaling pathway, HIF-1 signaling pathway, and pentose and glucuronate interconversions. Moreover, in line with the circadian pathway enrichment results, 6-h TRFM robustly inhibited cell proliferation and induced cell apoptosis and cell cycle arrest in lung adenocarcinoma A549 cells, lung adenocarcinoma H460 cells, esophageal carcinoma Eca-109 cells, and breast adenocarcinoma MCF-7 cells. ConclusionsOur findings provide the first in vitro mimicking medium for TRF intervention and indicate that 6-h TRFM is sufficient to reprogram the circadian signatures of lung adenocarcinoma cells and inhibit the progression of multiple tumors.

Inhibition of APLN suppresses cell proliferation and migration and promotes cell apoptosis in esophageal cancer cells in vitro, through activating PI3K/mTOR signaling pathway.

Esophageal cancer is the sixth leading cause of cancer mortalities globally with a high incidence rate. Apelin (APLN) plays regulatory roles in different organs. However, its role in esophageal cancer remains unknown. Therefore, our study aims to explore the effect of APLN on esophageal cancer. One hundred and eighty-four (184) esophageal tumor tissues samples from patients with esophageal cancer, and 11 esophageal tissues samples from healthy volunteers were analyzed for the expression of APLN. APLN was highly expressed in the tumor of patients with esophageal cancer and esophageal cancer cells. Patients with high expressions of APLN had a lower survival rate than the ones with low to medium expressions of APLN. Human esophageal carcinoma cell lines, TE-1 and ECA-109 cells were transfected with APLN siRNA to knockdown APLN, or transfected with pcDNA-APLN to overexpress APLN. Inhibition of APLN by siRNA-APLN reduced proliferative, migrative, and invasive abilities of esophageal cancer cells and promoted cell apoptosis, which could be all restored by pcDNA-APLN. Moreover, knocking down APLN by siRNA-APLN suppressed the PI3K/mTOR signaling pathway. These findings identify that APLN inhibition might ameliorate esophageal cancer through activating the PI3K/mTOR signaling pathway, thus APLN could be a potential target for esophageal cancer.

Retracted] Talen‑mediated girdin knockout downregulates cell proliferation, migration and invasion in human esophageal carcinoma ECA109 cells.

Following the publication of this paper, it was drawn to the Editors' attention by a concerned reader that the Transwell cell migration data shown in Fig.6 were strikingly similar to data appearing in different form in other articles by different authors; furthermore, there were other possible anomalies associated with these data. Owing to the fact that the contentious data in the above article had already been published elsewhere, or were already under consideration for publication, prior to its submission to Molecular Medicine Reports, the Editor has decided that this paper should be retracted from the Journal. After having been in contact with the authors, they agreed with the decision to retract the paper. The Editor apologizes to the readership for any inconvenience caused. [the original article was published in Molecular Medicine Reports 10: 848‑854, 2014; DOI: 10.3892/mmr.2014.2268].

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.

Lentiviral-Mediated Beclin-1 Overexpression Inhibits Cell Proliferation and Induces Autophagy of Human Esophageal Carcinoma Eca109 Cell Xenograft in Nude Mice.

Esophageal carcinoma is one of the common malignant tumors in digestive tract. BECLIN-1 is a key gene that regulates autophagy, and its abnormal expression may be related with many human tumors. However, the mechanism of BECLIN-1 in esophageal carcinoma remains unknown. In this study, we explored the effect of BECLIN-1 overexpression on tumor growth in mice with esophageal carcinoma and its mechanism. Recombined lentiviral vector containing BECLIN-1 was used to transfect human esophageal carcinoma Eca109 cells and establish stable cell line. qRT-PCR was used to detect BECLIN-1 mRNA level in the transfected Eca109 cells, CCK-8 assay was used to detect cell proliferation. Beclin-1, P62 and LC3-II protein expression levels in Eca109 cells were detected using Western blot analysis. Subcutaneous xenograft nude mice model of human esophageal carcinoma was established, and the tumor growths in Beclin-1 group, control group and empty vector group were monitored. Beclin-1 protein expression in vivo was detected by immunohistochemistry. Beclin-1 mRNA and protein were overexpressed in Eca109 cells. Compared with empty vector group, the growth rate of cells transfected with BECLIN-1 decreased significantly. Compared with the control group and empty vector group, the expression level of P62 protein in beclin-1 group was significantly decreased, while the expression level of LC3-II protein was significantly increased. The tumor growth rate in nude mice of Beclin-1 group was significantly lower than that of the control group and empty vector group, and Beclin-1 protein was mainly expressed in Beclin-1 group in vivo. BECLIN-1 can induce autophagy in esophageal carcinoma Eca109 cells, and it can significantly inhibit the growth of esophageal carcinoma.

Silence of cZNF292 suppresses the growth, migration, and invasion of human esophageal cancer Eca-109 cells via upregulating miR-206.

Circular RNA (circRNA) cZNF292 has been previously revealed as a circular oncogenic RNA. This study attempted to illustrate the functions of cZNF292 in human esophageal carcinoma Eca-109 cells. Eca-109 cells were transfected with the short hairpin RNA specific against cZNF292 (sh-cZNF292) and/or miR-206 inhibitor. cZNF292 and miR-206 expression was examined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Cell counting kit-8 and flow cytometry were performed for detecting cell growth including cell viability as well as apoptosis. Various kinds of factors, which are involved in cell development including proliferation, apoptosis, migration, and invasion were determined by western blot analysis. Besides, the activation of AMP-activated protein kinase (AMPK) and PI3K/AKT signaling was measured by western blot analysis. It was found that cZNF292 silencing decreased Eca-109 cell viability and induced apoptosis. In the meantime, cZNF292 silencing inhibited cell migration and invasion. cZNF292 silencing upregulated miR-206 expression. And miR-206 downregulation impaired the suppressive effects of cZNF292 silence toward Eca-109 cell growth, migration, and invasion. cZNF292 silencing activated AMPK signaling and inactivated PI3K/AKT signaling also via regulating miR-206. In conclusion, silencing of cZNF292 abated growth, migration, and invasion of Eca-109 cells by upregulating miR-206, which subsequently modulated AMPK and PI3K/AKT signaling pathways.

Effects of long noncoding RNA (linc-VLDLR) existing in extracellular vesicles on the occurrence and multidrug resistance of esophageal cancer cells

ObjectiveTo investigate the relationship between the expression of linc-VLDLR in extracellular vesicles (EVs) and esophageal carcinomas development and drug resistance. MethodsThe expression of linc-VLDLR and ABCG2 mRNA in 60 cases of esophageal carcinoma tissue, para-carcinoma tissue and the normal esophagus tissue were detected using Fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR). Fifty percent inhibiting concentration (IC50) of adriamycin (ADM) to Eca109 cells was detected by MTT assay, after the treatment of different concentrations of adriamycin (ADM) on esophageal squamous cell carcinoma Eca109 cell line for 24 h. EVs were extracted from culture medium after the treatment of three concentrations of ADM (setting based on the IC50) on Eca109 cells for 24 h. Linc-VLDLR expression in EVs was detected by qRT-PCR. After the treatment of the extracted EVs on virgin Eca109 cells for 48 h, then intervening these cells for 24 h by different concentrations of ADM, the new values of IC50 were detected by MTT assay. Cell cycle, cell apoptosis and ABCG2 protein expression of these Eca109 cells were detected by flow cytometry (FCM). Linc-VLDLR and ABCG2 mRNA expression in these Eca109 cells were detected by qRT-PCR. ResultsExpression of linc-VLDLR and ABCG2 mRNA in esophageal squamous cell carcinoma tissue were significantly higher than that in esophageal atypical hyperplasia and normal esophagus tissue, P < 0.01. After the treatment of ADM on Eca109 cells for 24 h, IC50 of Eca109 cells was detected as (0.44 ± 0.02) μg/mL, thus ADM concentrations of 0, 0.2, 0.4 and 0.8 μg/mL were selected to accomplish the following parts of this study. After four groups of Eca109 cells were treated by ADM in different concentrations separately, extracted EVs from the supernatant of all four groups, then labeling these four groups as EVs1, 2, 3 and 4. Linc-VLDLR expression in EVs4 was significantly higher than that in EVs1-3, P < 0.01. After the treatment of EVs1-4 on virgin Eca109 cells for 48 h, new values of IC50 of Eca109 to ADM were detected by MTT. It was found that the IC50 value of group EVs4 was significantly higher than that of other groups, P < 0.05. Flow cytometry results showed that the proliferation index of Eca109 cells in EVs4 was significantly higher than that in EVs1-3 and control groups, P < 0.01. Whereas, there was an obviously downward trend in the apoptosis rate of EVs4, compared to other three groups, P < 0.01. Linc-VLDLR and ABCG2 mRNA and protein expression level in Eca109 cells of EVs4 group were significantly higher than that of EVs1-3 and control groups, P < 0.05. ConclusionsHigh expression of Linc-VLDLR and ABCG2 gene in esophageal cancer cells affected the formation of esophageal cancer drug resistance. EVs released by drug-resistant cells were proved that they could upregulate the expression of ABCG2 in esophageal cancer cells and thus regulate the drug resistance of esophageal cancer cells, which was related to the linc-VLDLR carried by EVs.

Protective effect of propofol on hydrogen peroxide-induced human esophageal carcinoma via blocking the Wnt/β-catenin signaling pathway.

Objective(s):To analyze the potential influences of propofol on the oxidative stress of H2O2-induced human esophageal squamous cell carcinoma (ESCC) Eca109 cell through mediating the Wnt/β-catenin signaling pathway.Materials and Methods:Eca109 cells were classified into 5 groups: Control group, H2O2 group, Propofol + H2O2 group, Dkk1 (Dickkopf-1, Wnt/β-catenin pathway antagonist) + H2O2 group, and Propofol + LiCl (Lithium chloride, Wnt/β-catenin pathway agonist) + H2O2 group. Western blotting was performed to determine the protein expressions, flow cytometry to measure the content of ROS, immunofluorescence staining to detect the oxidative DNA damage, as well as MTT, AnnexinV-FITC/PI, Wound-healing, and Transwell assays to test the biological characteristics of Eca109 cells.Results:H2O2 resulted in the increased nuclear and cytoplasmatic expression of β-catenin, reduced p-GSK3β expression, up-regulated ROS content, and induced oxidative DNA damage in Eca109 cells. Moreover, Eca109 cells treated with H2O2 alone had enhanced cell proliferation and metastasis but decreased cell apoptosis, as compared with those without any treatment; meanwhile, the declined Cyt C, Bax, and cleaved caspase-3, as well as the elevated Bcl-2 were also observed in Eca109 cells in the H2O2 group, which were reversed by Propofol or Dkk1. Moreover, Propofol could inhibit the effect of LiCl on activating the Wnt/β-catenin signaling pathway in H2O2-induced Eca109 cells.Conclusion:Propofol elicits protective effects to inhibit H2O2-induced proliferation and metastasis and promote apoptosis of Eca109 cells via blocking the Wnt/β-catenin pathway, offering a possible therapeutic modality for ESCC.

Simvastatin, but not pravastatin, inhibits the proliferation of esophageal adenocarcinoma and squamous cell carcinoma cells: a cell-molecular study

Background and objectiveLong-term statin therapy has been shown to protect against several cancers, including esophageal cancer (EC). While the mechanisms underlying this effect are not clear. We investigated the effect of hydrophobic simvastatin and hydrophilic pravastatin on the proliferation of EC cells and sought to explore the underlying mechanisms.MethodsEsophageal adenocarcinoma OE-19 cells and esophageal squamous cell carcinoma Eca-109 cells were treated with different concentrations of simvastatin or pravastatin for 24 h and 48 h. Cell proliferation was assessed by Cell Counting Kit-8 assay. Malondialdehyde (MDA) levels were measured by thiobarbituric acid (TBA) assay. mRNA and protein expression of COX-2 were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot, respectively; The expression of prostaglandin E2 (PGE2) was measured by ELISA.ResultsSimvastatin, but not pravastatin, significantly inhibited the proliferation of OE-19 and Eca-109 cells in a dose- and time-dependent manner, accompanying with the increasing of the MDA level. Moreover, simvastatin suppressed the expression of COX-2 and PGE2 in both OE-19 and Eca-109 cells in a dose-dependent manner.ConclusionsLipophilic simvastatin, but not hydrophilic pravastatin, had significant inhibitory effects on the proliferation of Eca-109 and OE-19 cells. The reduction of COX-2 and PGE2 by simvastatin suggested that the inhibitory effect of simvastatin on the proliferation of EC cells may be independent of its lipid-lowering effect. Simvastatin may be a promising agent for the prevention and treatment of EC.

Inhibitory effect of microRNA-455-5p on biological functions of esophageal squamous cell carcinoma Eca109 cells via Rab31.

The aim of the present study was to examine microRNA (miRNA or miR)-455-5p expression in esophageal squamous cell carcinoma (ESCC) at the tissue and cellular levels in order to elucidate its biological roles. A total of 60 patients with ESCC were enrolled in the present study and reverse transcription-quantitative polymerase chain reaction was used to measure the expression of miR-455-5p. ESCC Eca109 cells were transfected with miR-NC, miR-455-5p mimics or inhibitor and a Cell Counting Kit-8 assay was used to assess proliferation. To investigate the migration and invasion abilities of Eca109 cells, Transwell and Matrigel assays were performed. Western blotting was employed to measure Rab31 protein expression, while a rescue assay was utilized to study the biological roles of miR-455-5p and Rab31 in Eca109 cells. To determine whether Rab31 is a direct target of miR-455-5p, a dual luciferase reporter assay was performed. The results revealed that miR-455-5p expression was decreased in ESCC tissues and was negatively correlated with metastasis and pathogenesis. In vitro overexpression of miR-455-5p inhibited the proliferation, migration and invasion of ESCC Eca109 cells. Furthermore, miR-455-5p regulated the expression of Rab31 protein in Eca109 cells. Rab31 overexpression promoted the proliferation, migration and invasion of Eca109 cells. Luciferase reporter assay results revealed that miR-455-5p is able to bind with the 3′-untranslated region of Rab31 mRNA to regulate its expression. In summary, the results of the present study suggest that miR-455-5p expression is decreased in ESCC tissues and is miR-455-5p is negatively correlated with lymphatic metastasis and differentiation. As a tumor-suppressor gene, miR-455-5p inhibits the proliferation, migration and invasion of ESCC Eca109 cells by suppressing the expression of Rab31.

The Curcumin Analogs 2-Pyridyl Cyclohexanone Induce Apoptosis via Inhibition of the JAK2-STAT3 Pathway in Human Esophageal Squamous Cell Carcinoma Cells.

Multiple modifications to the structure of curcumin have been investigated with an aim to improve its potency and biochemical properties. Previously, we have synthesized a series of curcumin analogs. In the present study, the anticancer effect of 2-pyridyl cyclohexanone, one of the curcumin analogs, on esophageal carcinoma Eca109 and EC9706 cell lines and its molecular mechanisms were investigated. 2-Pyridyl cyclohexanone inhibited the proliferation of Eca109 and EC9706 cells by inducing apoptosis as indicated by morphological changes, membrane phospholipid phosphatidylserine ectropion, caspase 3 activation, and cleavage of poly(ADP-ribose) polymerase. Mechanistic studies indicated that 2-pyridyl cyclohexanone disrupted mitochondrial membrane potential, disturbed the balance of the Bcl-2 family proteins, and triggered apoptosis via the mitochondria-mediated intrinsic pathway. In 2-pyridine cyclohexanone-treated cells, the phosphorylation levels of JAK2 and STAT3 were dose-dependently decreased and p38 and p-ERK signals were notably activated in a dose-dependent manner. Moreover, we found that the addition of S3I-201, a STAT3 inhibitor, led to a decreased expression level of Bcl-2 in Eca109 cells. The chromatin immunoprecipitation assay demonstrated that STAT3 bound to the promoter of Bcl-2 in the Eca109 cells. Furthermore, the mutation of four STAT3 binding sites (−1733/−1723, −1627/−1617, −807/−797, and −134/−124) on the promote of Bcl-2 gene alone attenuated the transcriptional activation of STAT3. In addition, down-regulation of STAT3 resulted in less of transcriptional activity of STAT3 on Bcl-2 expression. These data provide a potential molecular mechanism of the apoptotic induction function of 2-pyridyl cyclohexanone, and emphasize its important roles as a therapeutic agent for esophageal squamous carcinoma.

Mechanism of Oxymatrine-induced Human Esophageal Cancer Cell Apoptosis by the Endoplasmic Reticulum Stress Pathway.

Endoplasmic reticulum stress is one of the mechanisms of cell apoptosis. In this study, the mechanism of oxymatrine-induced human esophageal cancer Eca-109 cell apoptosis by the endoplasmic reticulum stress pathway was investigated. Eca-109 cells were cultured in vitro with different doses of oxymatrine (0.5, 1, 2 μg/mL) for 48 h. The cell viability and proliferation inhibition rate were examined by MTT assay and cell cycle assay. The apoptosis rate was examined by Annexin V-FITC/propidium iodide assay. The expression of endoplasmic reticulum stress markers, including binding immunoglobulin protein and CCAAT-enhancer-binding protein homologous protein, were determined by real-time quantitative polymerase chain reaction and western blotting, respectively. MTT data showed that oxymatrine significantly inhibited the proliferation of Eca-109 cells. The cell apoptosis rate was quantified by flow cytometry. The expression of binding immunoglobulin protein was markedly downregulated in oxymatrine-treated Eca-109 cells while that of CCAAT-enhancer-binding protein homologous protein was upregulated. Oxymatrine inhibited proliferation and induced apoptosis of human esophageal carcinoma Eca-109 cells. Thus, oxymatrine may be a potential agent for treating human esophageal cancer.

Bufalin induces apoptosis in human esophageal carcinoma ECA109 cells by inhibiting the activation of the mTOR/p70S6K pathway.

The present study examined whether bufalin could induce human esophageal carcinoma ECA109 cells apoptosis via inhibiting the activation of mechanistic target of rapamycin (mTOR)/p70 S6 kinase (p70S6K) pathway is discussed in this article. The present study used the esophageal squamous cell carcinoma ECA109 cell line to assess the apoptosis-inducing effects of bufalin via inhibition of the mTOR/p70S6K pathways. A plasmid containing the wild-type mTOR gene (wtmTOR) was transfected into ECA109 cells. The levels of p70S6K, phosphorylated (p)-p70S6K, cellular inhibitor of apoptosis-1 (cIAP-1) and Bcl-2-associated death promoter (BAD) in ECA109 cells were examined by western blot analysis, and apoptosis was detected by flow cytometry analysis and Giemsa staining. The results revealed that the expression of p-p70S6K was increased as the time progressed (at 0, 12 and 24 h), and then decreased at 30, 36, 42 and 48 h after transfection. The expression of cIAP-1 was significantly decreased as time progressed following the addition of bufalin, whereas that of BAD was increased. The levels of p-p70S6K and cIAP-1 were significantly higher in the wtmTOR-transfected group than in the control and empty vector-transfected groups, and then reduced following addition of bufalin; however, BAD expression was significantly lower in the wtmTOR-transfected group. The results of flow cytometry revealed the cell cycle of ECA109 was arrested at G2/M phase and the apoptotic rate was significantly lower in the wtmTOR-transfected group than in the control and empty vector-transfected groups, and then increased following addition of bufalin. In conclusion, the findings of the present study demonstrated that bufalin induced apoptosis in esophageal carcinoma cells via the inhibition of the mTOR/p70S6K pathway and indicated that treatment with bufalin could be combined with chemotherapy to overcome the resistance of esophageal carcinoma cells to chemotherapeutic-induced apoptosis.

Down-regulation of STAT3 induces the apoptosis and G1 cell cycle arrest in esophageal carcinoma ECA109 cells

BackgroundSignal transducer and activator of transcription 3 (STAT3) is persistently activated in a wide variety of epithelial cancers. Aberrant activity of STAT3 correlates with tumor growth, invasion and metastasis, which makes it a potential therapeutic target of cancer. To explore the biological role of STAT3 in esophageal cancer, we used small hairpin RNA to knockdown the expression of the STAT3 gene in the esophageal carcinoma ECA109 cell line and the cell apoptosis, cell cycle and cell migration were investigated.MethodsThe cell apoptosis was tested using DNA ladder, mitochondrial membrane potential assay, TUNEL assay, annexin V-PI staining. Cell cycle phases were estimated using flow cytometry analysis. The mRNA and proteins related to apoptosis and cell cycle were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. And cell migration was investigated by in vitro Transwell assay. The data were analyzed with two-sample Student’s t test and ANOVA followed by the LSD post hoc test.ResultsOur results showed that knockdown of STAT3 in ECA109 cells induced noticeable apoptotic morphological changes like cell shrinkage, apoptotic vacuoles, membrane blebbing time-dependently. In addition, DNA ladder, TUNEL assay, Annexin V-PI staining and declined level of cleaved Caspase-3 indicated that down-regulation of STAT3 could induce apoptosis in ECA109 cells. Flow cytometry analysis displayed the induction of G1-phase cell cycle arrest of ECA109 cells by STAT3 decreasing, consistent with the descend of c-Myc and cyclin D1 in protein levels. Furthermore, STAT3 knockdown suppressed the expression of matrix metalloproteinases-9, sushi domain containing 2 and urokinase plasminogen activator in ECA109 cells and inhibited cell migration ability.ConclusionsKnockdown of STAT3 could induce the apoptosis and G1 cell cycle arrest in esophageal carcinoma ECA109 cells, and inhibit the migration ability of cells as well.

Celastrus orbiculatus extracts induce cell cycle arrest and apoptosis in human esophageal squamous carcinoma ECA-109 cells in vitro via the PI3K/AKT/mTOR signaling pathway.

Recently, Celastrus orbiculatus ethyl acetate extracts (COE) have been investigated for their anticancer effects on digestive tract tumors. However, the therapeutic effects of COE on esophageal squamous carcinoma cells (ESCC) have not been investigated. In the present study, the effects of COE on the cell cycle and apoptosis of ESCCs were assessed in vitro, and it was revealed that COE treatment triggered G0/G1 cell cycle arrest, and induced DNA damage and apoptosis in a dose-dependent manner in ESCC. Activation of the phosphatidylinositol 3-kinase/protein kinase-B/mechanistic target of rapamycin (mTOR) pathway was also suppressed by COE. Additionally, the combined treatment with COE and rapamycin (an mTOR inhibitor) acted synergistically in ECA-109 cells compared with the treatment with COE or rapamycin alone. These findings extend the understanding of the action of COE and suggest that COE has potential as a treatment option for ESCC as a single treatment or in combination.

Effect of lentivirus-mediated expression of RNF2 gene on the proliferation and migration of esophageal carcinoma cells following X-ray radiotherapy

Objective To examine the effect of X-ray radiotherapy on cell proliferation, migration, apoptosis, and cell cycle of human esophageal carcinoma ECA-109 cells following RNA interference (RNAi)-mediated downregulation of RNF2 gene expression. Methods The level of RNF2 mRNA expression in the human esophageal carcinoma cell line ECA-109 was determined using RT-PCR. Cell proliferation of ECA-109 was measured by MTT assay, and the changes in RNF2 protein expression in ECA-109-R cells were determined using Western blot. The changes in cell cycle and cell apoptosis at different time points following radiation were analyzed by flow cytometry, and the effect of transduction on cell migration was examined using Transwell migration assay. Data were subjected to an analysis of variance with repeated measurement design. Results The mean mRNA and protein levels of RNF2 in ECA-109 cells were significantly increased in a dose-dependent manner in the radiation group than in the control group (P 0.05). The Transwell migration assay showed that the number of migrating cells following 3.5 h of radiation was significantly lower in the ECA-109-R group than in the ECA-109 and ECA-109-N groups (P<0.01). The percentage of G2/M phase cells in each group was significantly lower following 6 Gy radiation compared to that in the corresponding untreated group (P<0.01), and the percentage of G2/M phase cells was significantly lower in the ECA-109-R group than in the ECA-109 and ECA-109-N groups (P<0.05). Furthermore, cell apoptosis following radiation was also significantly higher in the ECA-109-R group than in the ECA-109 and ECA-109-N groups (P<0.01). Conclusions RNAi-mediated downregulation of RNF2 expression in esophageal carcinoma cells can reduce cell proliferation and cell migration, rescue post-radiation G2/M cell cycle arrest, promote cell apoptosis, and increase radiosensitivity. Key words: RNF2 gene; Cell proliferation; Apoptosis; Cell cycle; Esophageal carcinoma cell line

Effects of bufalin on the mTOR/p70S6K pathway and apoptosis in esophageal squamous cell carcinoma in nude mice.

The aim of this study was to investigate the effects of bufalin on the mammalian target of rapamycin (mTOR/p70S6 kinase (p70S6K) signaling pathway and cell apoptosis in orthotopically transplanted tumors in nude mice. The mice were inoculated with human esophageal squamous cell carcinoma (ESCC) ECA109 cells in order to establish a model of orthotopicall transplanted ESCC tumors. The mice are administered low, medium and high doses of bufalin (0.5, 1.0 and 1.5 mg/kg) or rapamycin, or a combination of both. After the tumors were removed, the mRNA expression levels of mTOR, p70S6K, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), cellular inhibitor of apoptosis protein 1 (cIAP1) and caspase-3 were detected by RT-PCR. In addition, we performed western blot analysis and immunohistochemical analysis to determine the protein expression of mTOR, p70S6K, 4EBP1, cIAP1, active caspase-3, Bcl-2 and Bad in the tumor tissue. The results revealed that bufalin exerted a significant anti-tumor effect in the nude mice with ESCC orthotopically transplanted tumors. This was shown by the decrease in the expression of mTOR, p70S6K and 4EBP1, which suggested that bufalin may possibly be used to inhibit tumor growth via the inhibition of the activation of p70S6K and 4EBP1. We also found that bufalin decreased the expression of cIAP1 and Bcl-2, and increased that of active caspase-3 and Bad, thus indicating that bufalin promoted apoptosis. Thus, our findings suggest that bufalin promotes tumor cell apoptosis, and this may be one of the important anti-tumor mechanisms of action of bufalin.

Nestin expression involves invasiveness of esophageal carcinoma and its downregulation enhances paclitaxel sensitivity to esophageal carcinoma cell apoptosis.

Paclitaxel has been generally used to treat primary and metastatic esophageal carcinoma. It has been shown that nestin is highly expressed in esophageal carcinoma and that there is a strong association of nestin expression with poor prognosis in esophageal carcinoma patients. In this study, using immunohistochemistry, in situ hybridization and Western blotting we demonstrated that nestin was overexpressed in the invasive esophageal carcinoma. To further elucidate whether nestin inhibition could enhance paclitaxel sensitivity to esophageal carcinoma cells, we applied nestin siRNA in esophageal squamous cell carcinoma Eca-109 cells. Flow cytometry and TUNEL staining both showed that combination of paclitaxel treatment and nestin knockdown resulted in greater induction of apoptosis of esophageal carcinoma cells as compared with the cells transfected with control siRNA (also treated with paclitaxel). This study indicates that nestin knockdown enhances chemotherapeutic sensitivity of paclitaxel to esophageal carcinoma, and suggests that silencing of nestin could be a valuble therapeutic approach for enhancing drug sensitivity and thereby improving the treatment outcome of esophageal carcinoma patients.

Effects and its mechanism of Nimotuzumab on radiosensitivity of esophageal carcinoma ECA-109 and TE-13 cell lines

Objective: To investigate the effects of nimotuzumab on radiosensitivity of ECA-109 and TE-13 esophageal carcinoma cell lines and explore its possible mechanism. Methods: The ECA-109 and TE-13 cells were divided into control group, irradiation group, medicine group, and combined group (irradiation + medicine). In the combined group, ECA-109 and TE-13 cells were treated with nimotuzumab for 24 h before irradiation, and the cells were collected 2 h after irradiation. The radiosensitizing effects of nimotuzumab on ECA-109 and TE-13 cells were evaluated by clone formation assay. Cell apoptosis was detected by flow cytometry. Western blotting was used to evaluate the expression of EGFR, p-EGFR, DNA-PKcs, p-DNA-PKcs and γH2AX. Results: The values of Dq (quasithreshold dose), D0(mean lethal dose)and SF2 (surviving fraction at 2 Gy) of ECA-109 and TE-13 cells in the combined group were significantly lower than those of the radiation group (for ECA-109 cells, 1.11 vs. 1.72, 1.40 vs. 2.14, 0.42 vs. 0.66, respectively; for TE-13 cells, 0.41 vs. 0.46, 0.43 vs. 0.65, 0.40 vs. 0.71, respectively (all P<0.05). The sensitivity enhancement ratio (SER) of ECA-109 and TE-13 cells were 1.35 and 1.43, respectively. Flow cytometry showed that the apoptosis rate of ECA-109 and TE-13 cells in the combined group were significantly higher than those of the radiation group [for ECA-109 cells, (41.31±1.52)% vs. (9.54±0.52)%; for TE-13 cells, (46.28±0.28)% vs. (11.32±0.31)%, both P<0.01]. Western blotting showed that the expression levels of EGFR and DNA-PKcs were not significantly different in all groups (all P>0.05). Compared with those of the control group, p-EGFR and p-DNA-PKcs of the radiation group were significantly higher in both cell lines (P<0.05), and the γH2AX levels in the radiation group and medicine group were significantly higher than that of the control group (P<0.05). Compared with those of the radiation group and medicine group, p-EGFR and p-DNA-PKcs protein expression in the combined group were decreased significantly (P<0.05), while γH2AX protein expression was significantly increased (P<0.05). Conclusions: Nimotuzumab can enhance the radiosensitivity of esophageal cancer ECA-109 and TE-13 cells. The potential mechanism may be related to the inhibition of EGFR phosphorylation and down-regulation of DNA damage repair proteins. The radiosensitizing effect of nimotuzumab is greater on poorly differentiated esophageal cancer cells.

Effect of RAD18-siRNA on proliferation and chemotherapy sensitivity of human esophageal squamous cell carcinoma ECA-109 cells

Objective: To investigate the effect of RAD18-siRNA on cell proliferation and chemotherapy sensitivity of esophageal squamous cell carcinoma (ESCC) ECA-109 cells. Methods: RAD18-siRNA was transfected into human ECA-109 cells by Lipofectamine 3000. Quantitative PCR and Western blot were performed to detect RAD18 and CyclinD1 expression; CCK-8 assay was used to determine cell proliferation and chemotherapy drug sensitivity; flow cytometry was used to determine cell cycle. Correlation between RAD18 and CyclinD1 mRNA expression was analyzed by Pearson's correlation. Results: Compared with non-transfected cells, the expression of RAD18 in RAD18-siRNA group was significantly decreased (P<0.05). The cell proliferation was inhibited (P<0.05) and the cell number of G1 phase was increased, G2/M phase cells decreased (P<0.05) in RAD18-siRNA group. After treatment with different concentrations of cisplatin or 5-FU, the survival rate of the two cell groups was reduced (all P<0.05), and the IC50 of RAD18-siRNA group was significantly lower than that of non-transfected group (P<0.05). The mRNA expression of RAD18 was positively correlated with CyclinD1 expression in ESCC tissues(r=0.478, P<0.01). Conclusion: Down-regulated expression of RAD18 can decrease the cell proliferation and increase chemo-sensitivity of ESCC cells, and CyclinD1 may participate in the process.