Esophageal Carcinoma EC9706 Cells Research Articles

Inhibition of PDK1 gene expression in esophageal cancer EC9706 cells by RNA interference and its effect on their malignant biological behavior

To explore the silencing effects of RNA interference on the expression of 3-phosphoinositide-dependent protein kinase 1 (PDK1) gene, and the effects on malignant phenotypes of esophageal carcinoma EC9706 cells. PDK1 siRNAs was transfected into the EC9706 cells. The expression of PDK1 mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR). At the same time, expressions of PDK1, Akt and phosphorylated Akt proteins were detected by Western blot. Methyl thiazolyl tetrazolium assay (MTT) was used to examine the cell proliferation after transfection. Flow cytometry was used to determine the percentage of apoptosis cells, and Transwell chambers were used to detect the invasion ability of the cells. Tumor formation in nude mice was used to assess the tumorigenic characteristics in vivo. Compared with the non-transfected group, PDK1 siRNA effectively inhibited the expression of PDK1 mRNA in EC9706 cells, with an inhibition rate of (28.5 ± 4.2)% at 24 h, (51.1 ± 5.7)% at 48 h and (60.6 ± 4.1)% at 72 h after transfection. The expressions of PDK1 and phosphorylated Akt protein were also knocked down by PDK1 siRNA (P < 0.05). PDK1 siRNA significantly inhibited the cell proliferation and invasion, promoted the cell apoptosis, and inhibited the EC9706 cells proliferation in vivo and the expression of PDK1 protein in the transplanted tumors (P < 0.05). PDK1 may play an important role in esophageal cancer cell proliferation, invasion and apoptosis, and may serve as an effective target for cancer gene therapy.

Potential transcriptional regulatory regions exist upstream of the human ezrin gene promoter in esophageal carcinoma cells

We previously demonstrated that the region -87/+134 of the human ezrin gene (VIL2) exhibited promoter activity in human esophageal carcinoma EC109 cells, and a further upstream region -1324/-890 positively regulated transcription. In this study, to identify the transcriptional regulatory regions upstream of the VIL2 promoter, we cloned VIL2 -1541/-706 segment containing the -1324/-890, and investigated its transcriptional regulatory properties via luciferase assays in transiently transfected cells. In EC109 cells, it was found that VIL2 -1541/-706 possessed promoter and enhancer activities. We also localized transcriptional regulatory regions by fusing 5'- or 3'-deletion segments of VIL2 -1541/-706 to a luciferase reporter. We found that there were three positive and one negative transcriptional regulatory regions within VIL2 -1541/-706 in EC109 cells. When these regions were separately located upstream of the luciferase gene without promoter, or located upstream of the VIL2 promoter or SV40 promoter directing the luciferase gene, only VIL2 -1297/-1186 exhibited considerable promoter and enhancer activities, which were lower than those of -1541/-706. In addition, transient expression of Sp1 increased ezrin expression and the transcriptional activation of VIL2 -1297/-1186. Other three regions, although exhibiting significantly positive or negative transcriptional regulation in deletion experiments, showed a weaker or absent regulation. These data suggested that more than one region upstream of the VIL2 promoter participated in VIL2 transcription, and the VIL2 -1297/-1186, probably as a key transcriptional regulatory region, regulated VIL2 transcription in company with other potential regulatory regions.

Characterization of apoptosis and proliferation in esophageal carcinoma EC109 cells following siRNA-induced down-regulation of TRAF6

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an activator of the NF-κB transcription factor. NF-κB is involved in a variety of inflammatory, anti-apoptotic, and gene regulatory pathways and was recently found to be over-expressed in esophageal cancer cells. Here we investigated the function of TRAF6 in the esophageal cancer cell line EC109. siRNA targeting TRAF6 was introduced into EC109 cells and TRAF6 mRNA and protein levels were subsequently examined via RT-PCR and western blotting. Rates of apoptosis and cell proliferation were also measured using flow cytometry, ethynyl deoxyuridine (EdU), and CCK-8 (Cell Counting Kit-8) assays. The real-time PCR array was applied to profile the expression of TRAF6 related genes. TRAF6-siRNA reduced TRAF6 mRNA and protein expressions. NF-κB p65 protein expression was decreased in TRAF6-targeting siRNA-transfected cells compared to cells of the negative control. TRAF6-siRNA also significantly inhibited proliferation and enhanced apoptosis of EC109 cells. These studies suggested that TRAF6 was required for NF-κB activation in EC109 cells and it may be a good molecular target for suppressing the survival and proliferation of esophageal cancer cells.

A Cyclooxygase-2 Inhibitor NS-398-Enhanced Apoptosis of Esophageal Carcinoma Cell EC9706 by Adjusting Expression of Survivin and Caspase-3

The aim of this study is to examine the effect and mechanism of a selective cyclooxygenase-2 (COX-2) inhibitor NS-398 on inducing apoptosis of esophageal cancer cells. After the treatment with NS-398 on esophageal carcinoma EC9706 cell, MTT assay was used to observe the inhibition of EC9706 cell growth and apoptosis was determined by electronic microscopy and flow cytometry. The expression of survivin and caspase-3 was examined using immunocytochemical technique. The dose of 0.01–0.1 mM NS398 showed the inhibitory effect on growth of EC9706 cell lines and induce apoptosis in a dose- and time-effective manner; moreover, NS-398 also downregulated the level of expression of survivin and elevated the expression of capase-3. NS-398 can induce apoptosis of the esophageal carcinoma EC9706 cells by means of adjusting expression of survivin and caspase-3.

Promoting effect of all-trans retinoic acid on the chemosensitivity of esophageal cancer EC9706 cells

To investigate the impact of all-trans retinoic acid (ATRA) on chemosensitivity to esophageal squamous cell carcinoma EC9706 cells in vitro and its mechanism. EC9706 cells were routinely cultured as the control group. The experimental group was divided into three groups. The ATRA group with ATRA in final concentration of 1 µmol/L; the 5-Fu group with 5-Fu in final concentration of 50 mg/L; the combined treatment group with ATRA in final concentration of 1 µmol/L and 5-Fu 50 mg/L. The cell apoptosis was detected by terminal deoxynucleotidy transferase mediated dUTP nick end labelling (TUNEL). The cell cycle and apoptosis were detected by flow cytometry. The results of TUNEL showed that in the combined treatment group appeared a large number of apoptotic cells, and their nuclei were stained brown, with a positive rate of 89.7%. There was a significant difference in the comparison with the ATRA group (38.3%) and 5-Fu group (40.3%) (P < 0.05). The flow cytometry showed that the ATRA + 5-Fu group had a significantly higher apoptosis rate (76.9% ± 2.7%) than that in the ATRA group (38.2% ± 2.6%) and 5-Fu group (45.2% ± 2.3%) (P < 0.05). The ratio of cells in G(1) phase increased in the ATRA + 5-Fu group (83.4% ± 3.0%), significantly higher than (48.2% ± 2.5%) in the ATRA group and (53.2% ± 2.6%) in the 5-Fu group (P < 0.05). The ratio of cells in S + G(2)/M phase was decreased in the ATRA + 5-Fu group, with a significant difference (P < 0.05) when compared with other groups. There was no significant difference between the ATRA group and 5-Fu group (P > 0.05) in the apoptosis rate and the proportion of cells at different phases. ATRA can induce apoptosis of esophageal carcinoma EC9706 cells in vitro. The combination of ATRA and 5-Fu may enhance the chemotherapeutic efficacy.

Effect of gecko alcohol extract on human esophageal squamous carcinoma cell line EC9706 and anti-tumor activity in vivo

To observe the anti-tumor activity of gecko alcohol extract in vitro and in vivo. in vitro, the inhibitory effect of gecko alcohol extract on proliferation of human esophageal carcinoma EC9706 cells was measured by MTT colorimetric assay. Morphological change of apoptotic cells was observed by Hoechst33258 fluorescence staining and apoptosis induced by gecko alcohol extract was evaluated in TUNEL assay. The expression of apoptosis protein Bax and Bcl-2 in EC9706 cells was investigated by immunohistochemistry. in vivo, the inhibitory effect of gecko alcohol extract on tumor growth was examined on S180 sarcoma model. After gecko alcohol extract (6-8 g x L(-1)) treatment for 24, 48 and 72 h separately, EC9706 cell proliferation was significantly inhibited in both dose- and time- dependent manner (P < 0.01). Cell apoptosis in gecko alcohol extract-treated group (6, 7 g x L(-1)) was significantly higher than that in control group [(12.73 +/- 3.84)%, (9.80 +/- 2.32)% vs. (5.87 +/- 2.54)%, P < 0.05, P < 0.01]. Although the level of Bcl-2 did not change significantly, the expression of Bax was rembarkably up-regulated in gecko alcohol extract treated group. Gecko alcohol extract inhibited the growth of S180 sarcoma in Kun-ming mice at all doses (0.6, 1.2, 2.4 g x kg(-1)) of administration. The inhibitory rate was 44.88%, 63.94% and 69.53% respectively. From tumor inhibitory test, gecko alcohol extract shows significantly inhibitory effect in vitro and in vivo. The increased Bax/Bcl-2 ratio is mechanistically linked to the gecko alcohol extract-induced tumor cell apoptosis.

Up-regulation of stathmin induces growth arrest of esophageal squamous cell carcinoma EC9706 cell

To construct a eukaryotic expression vector of stathmin gene and investigate its effect on esophageal squamous cell carcinoma (ESCC) EC9706 cell line. Stathmin cDNA coding sequence was amplified by RT-PCR and cloned into a eukaryotic expression vector pcDNA3.1(+). EC9706 cells were transfected with this recombinant plasmid pcDNA3.1-stathmin by lipofectamine. And the stable transfectants were selected with G418 medium. Stathmin protein expression was detected with Western blot in transfected EC9706 cell lines. Morphologic change of stable transfectants was observed under microscope. The proliferation of transfected cells was measured by cell counting, MTT and in vitro formation assay of flat. Flow cytometry was used to detect the cell cycle. Nude mice were adopted to investigate the in vivo tumorigenic characteristics of the transfected cells. A 450 bp coding sequence of stathmin cDNA was amplified by RT-PCR and then cloned into pcDNA3.1(+) plasmid to harvest the recombinant plasmid pcDNA3.1-stathmin. The recombinant plasmid pcDNA3.1-stathmin and blank vector were transfected respectively into EC9706 cells. The up-regulated expression of stathmin protein was validated by Western blot (P < 0.01). Compared with control, EC9706 cells transfected with pcDNA3.1-stathmin appeared swollen and multi-nuclear with a cell mitotic arrest; doubling generation time of pcDNA3.1-stathmin transfectants was prolonged (25 - 28 h); The in vitro cell proliferation ability and clone formation rate (34.5% ± 6.9%) decreased, cell cleavage was blocked at G(2)/M phase (21.7% ± 3.4%) and the oncogenicity of inoculated cells in nude mice decreased (all P < 0.01). The up-regulated expression of stathmin protein triggered by the recombinant plasmid pcDNA3.1-stathmin can inhibit the proliferation and oncogenicity of ESCC EC9706 cells. This molecule may be a promising therapeutic target in ESCC patients.

Expression of Bmi-1 gene in esophageal carcinoma cell EC9706 and its effect on cell cycle, apoptosis and migration

Previous studies have shown that Bmi-1 is overexpressed in a variety of tumors, suggesting that Bmi-1 plays an important role in tumorigenesis. In this study, we investigated the effect of Bim-1 siRNA on cell proliferation, cell cycle, cell apoptosis and migration of human esophageal carcinoma EC9706 cells, and explored its potential mechanisms. Bmi-1 small interfering RNA (siRNA) was transferred into EC9706 cells. Then, cell proliferation was measured using cell counting kit-8 (CCK-8), cell cycle and cell apoptosis were analyzed by flow cytometry, cell migration ability was detected using Boyden chamber assay, and the mRNA and protein expression levels of Bmi-1, p16, Bcl-2, Bax, and MMP-2 were determined using real-time polymerase chain reaction (PCR) and Western blot analysis, respectively. Bmi-1 siRNA treatment significantly inhibited the expression of Bmi-1 at both mRNA and protein levels in EC9706 cells. Cell proliferation rate decreased dramatically in the Bmi-1 siRNA treated group than in the untreated group and in the scrambled siRNA treated group (both P < 0.001). In Bmi-1 treated group, the percentage of cells at G(0)/G(1) stage was 71.93%, which was higher than that in the untreated group (47.36%) or scramble siRNA treated group (48.47%) (both P < 0.001). Early cell apoptosis rate also increased significantly in the Bmi-1 siRNA treated group (both 17.32%) than in the untreated group (2.61%) and in the scramble siRNA treated group (2.73%) (both P < 0.001). Further experiment suggested that downregulation of Bmi-1 led to less cell migration. In EC9706 cells transfected by Bmi-1 siRNA, the expression levels of p16 and Bax increased, while the expression level of Bcl-2 decreased. Bmi-1 downregulation in esophageal carcinoma cells inhibits cell proliferation, cell cycle, and cell migration, while increases cell apoptosis. These results suggest that Bmi-1 is a potential molecular target of treating esophageal cancer.

Epidermal growth factor-induced epithelial–mesenchymal transition in human esophageal carcinoma cells – A model for the study of metastasis

Deciphering the molecular basis of esophageal cancer metastasis requires adequate experimental models. Epithelial-mesenchymal transition (EMT) is the hallmark of tumor metastasis. As a promoter of the malignant progression of esophageal cancer, epidermal growth factor (EGF) has been shown to induce EMT in several cell lines. In this study we examined the effects of EGF on esophageal carcinoma EC109 cells. We found that EGF at high concentration induced the cells to undergo morphological change, exhibit higher invasive and metastatic potential, as well as change in the expression of lineage markers. This EMT model might facilitate mechanistic studies of esophageal cancer metastasis.

Effect of human angiopoietin-like protein 4 overexpression on the growth of esophageal carcinoma EC9706 cells

The aim of the study was to construct the eukaryotic expression vector of human angiopoietin-like protein 4 (ANGPTL4) and observe the effect of ANGPTL4 overexpression on the growth of esophageal carcinoma EC9706 cells. Total RNA was extracted from normal hepatic tissue, and ANGPTL4 cDNA was amplified by RT-PCR. The PCR product was doubly digested by XbaI and SalI, and then recombined into eukaryotic expression vector. Then, pIRES-GFP-ANGPTL4 was obtained by G418 selection, then pIRES-GFP-ANGPTL4 and pIRES-GFP were transfected into EC9706 cells with lipidosome-packaged method. Meanwhile, the transfected cells were selected by G418, and then stable transfected cell lines were obtained. ANGPTL4 mRNA levels, the cell cycles and growth curves of EC9706 cells in experiment group (transfected with pIRES-GFP-ANGPTL4), empty vector group (transfected with pIRES-GFP) and blank control group (EC9706 cells without transfection) were detected with RT-PCR, flow cytometry and MTT methods, respectively. Eukaryotic ANGPTL4 expression vector pIRES-GFP-ANGPTL4 was successfully constructed. The ANGPTL4 mRNA level (0.21 ± 0.03) in experiment group was significantly higher than that of the empty vector group (0.04 ± 0.008) and the blank control group (0.05 ± 0.007), with significant differences (P < 0.01). The proportion of cells in S phase in experiment group was significantly different with those of the other two groups (P < 0.05). The cell growth of EC9706 cells in experiment group was slower than those of the other two groups. From the third day, the differences began to be significant. ANGPTL4 overexpression in esophageal carcinoma EC9706 cells could inhibit the growth of EC9706 cells.

Effects of paeonol and 5-FU on the proliferation and apoptosis of human esophageal carcinoma EC9706 cells

Effects of paeonol and 5-FU on the proliferation and apoptosis of human esophageal carcinoma EC9706 cells

Fascin overexpression is regulated by the transactivation of the promoter but not by its hypomethylation in esophageal squamous cell carcinoma

Fascin 1 (fascin) is known to be overexpressed in esophageal squamous cell carcinoma (ESCC); however, the mechanisms of this overexpression are unclear. In this study, the FSCN1 core promoter was isolated and the transcriptional regulatory mechanism of fascin overexpression in ESCC was investigated. By combining the use of progressive 5' deletions and dual-luciferase reporter assays, the FSCN1 core promoter was identified within the -74/-41 region in esophageal carcinoma EC109 cells harboring a GC box and a composite CRE/AP-1 binding site. Further analysis demonstrated that only the GC box was essential for transcription. No methylated CpG sites were found within the FSCN1 promoter in normal and tumor cells or tissues examined by methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS), which suggested that hypomethylation did not contribute to the overexpression of fascin in ESCC. Furthermore, the region of +168/+2838 was found to inhibit promoter activity. Additional BGS analysis indicated that the region of +560/+859 (in exon 1) was hypermethylated in normal and tumor cells or tissues. Unexpectedly, demethylation did not eliminate the suppression, suggesting that a silencer caused this suppression, and not DNA methylation in exon 1. Taken together, the results indicate that fascin overexpression in ESCC is regulated by the transactivation of the fascin promoter, but not by its hypomethylation.

Isolation and Identification of Cancer Stem-Like Cells in Esophageal Carcinoma Cell Lines

Side population (SP) cells may play an important role in tumorigenesis and cancer therapy. We isolate and identify the cancer stem-like cells in human esophageal carcinoma (EC) cell lines, EC9706 and EC109 cells labeled with Hoechst 33342. Both the cell lines contained SP cells, and the cells that had the strongest dye efflux activity ("Tip"-SP cell) in EC9706 had higher clone formation efficiency than non-SP cells. When transplanted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, "Tip"-SP cells showed at least 50 times higher tumorigenicity than non-SP cells. Microarray analysis discriminated a differential gene expression profile between "Tip"-SP and non-SP cells, which is further tested using quantitative real-time RT-PCR. It is ascertained that several important stem cell-related genes (including OCT-4, SOX-2, BMI-1, and ZFX), two ATP-binding cassette (ABC) transporter genes (ABCG2 and ABCA5), and three Wnt and two Notch signal pathway-related genes (such as FZD10, PTGS2, KLF5, TTK, and RBM15) were upregulated in "Tip"-SP cells. Western blot showed a higher expression of beta-catenin protein in "Tip"-SP cells. All these indicated that the minority population described as "Tip"-SP cells possessed cancer stem cell character. Further understanding of tumor stem cell-specific traits will offer insights on the early stages of tumorigenesis for prevention and enhanced selectivity of antitumor therapeutics.

Small interfering RNA-mediated downregulation of targeting protein for Xklp2 suppresses the proliferation of human esophageal carcinoma EC9706 cells

Small interfering RNA-mediated downregulation of targeting protein for Xklp2 suppresses the proliferation of human esophageal carcinoma EC9706 cells