Human Esophageal Cell Research Articles

Extracellular matrix affects different aspects of cell behaviour potentially involved in response to aminolevulinic acid-based photoinactivation

Two-dimensional cell cultures do not seem to be reliable models for anticancer drug discovery and validation. Numerous in vitro tumour models of different complexity have been evaluated with the aim to enhance anticancer drug development, but whether all these models could be considered as physiologically relevant is a question. Even type of the extracellular matrix may markedly influence experimental results and supposedly also clinical treatment outcome. By using three human oesophageal cell lines and three-dimensional cultures based on collagen type I, abundant component of stromal tissue, and Matrigel, a surrogate of basement membrane, we tested the impact of extracellular matrix on different aspects of cell behaviour. We applied live cell fluorescence confocal microscopy in combination with image analysis and supplemented it with immunohistochemical analysis of differentiation markers in fixed samples. We found that cell morphogenesis, differentiation, extracellular vesicle formation, protoporphyrin IX production from aminolevulinic acid and response to subsequent photodynamic intervention induced by red light may be affected by the type of extracellular matrix and these modifications occur in a cell-type dependent manner. Our results demonstrate that the choice of the correct extracellular matrix for in vitro tumour models is crucial for gathering clinically relevant information from in vitro experimental studies.

Apoptotic Effects of Cryptotanshinone on Human Esophageal Carcinoma HCE-4 Cells and Related Mechanism

Apoptotic Effects of Cryptotanshinone on Human Esophageal Carcinoma HCE-4 Cells and Related Mechanism

Expressions of a disintegrin and metalloprotease 17 and epidermal growth factor recepter in esophageal squamous cell carcinoma and their significances

Objective To investigate the expressions of a disintegrin and metalloprotease 17 (ADAM17) and epidermal growth factor recepter (EGFR) in human esophageal squamous cell carcinoma (ESC), and to explore their relationship with clinicopathological characteristics. Methods The paraffin specimens in postoperative pathological tissues of 66 ESC patients in the Third People's Hospital of Hefei from January 2013 to December 2017 were selected. Expressions of ADAM17 and EGFR proteins were examined by using immunohistochemistry in 66 cases of ESC tissues and 33 cases of adjacent tissues of the tumors. The relationship of ADAM17 and EGFR with clinicopathological features was analyzed. Kendall method was used to detect the expression correlation of ADAM17 and EGFR. Results The positive rate of ADAM17 protein in ESC tissues was higher than that in the adjacent tissues of the tumors [68.2 % (45/66) vs. 33.3 % (11/33), χ 2 = 10.874, P = 0.001]. The positive rate of EGFR protein in ESC tissues was higher than that in the adjacent tissues of the tumors [66.7 % (44/66) vs. 39.4 % (13/33), χ2 = 6.699, P = 0.01]. The expressions of ADAM17 and EGFR protein were related with ESC pathological TNM staging, infiltration depth, lymph node metastasis (χ2 = 4.797, 4.890, 6.089; 8.790, 8.766, 10.154, respectively, all P < 0.05). ADAM17 expression was positively correlated with EGFR protein (rs = 0.368, P < 0.05). Conclusions ADAM17 and EGFR are highly expressed in human ESC. Besides, ADAM17 and EGFR have the interaction in the occurrence and development of esophageal cancer. Joint detection may help to determine the degree of metastasis and evaluate prognosis. Key words: Esophageal neoplasms; A disintegrin and metalloprotease 17; Recepter, epidermal growth factor; Immunohistochemistry

Abstract 1043: Patient-derived and cell line xenograft growth in the B6;129-Rag2 tm1Fwa IL2rg tm1Rsky/DwlHsd (R2G2) mouse model

Abstract Immunodeficient mouse models are helping to advance the field of oncology. A new model on the market, the B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) knockout mouse, lacks responsiveness to common gamma chain cytokines, including IL-2, IL-4, IL-7, IL-9 and IL-15. In addition, this model exhibits defects in lymphoid development and so lacks mature lymphocytes of the B, T, and natural killer (NK) cell lineages. Herein we describe growth of multiple patient-derived (PDX) and tumor cell line xeno- (CDX) and allografts in the R2G2 immunodeficient mouse model. The PDXs examined include colorectal and head and neck cancers. The CDXs studied include esophageal (OE33 and FLO1) and stomach cancer (AGS). Tumor growth data were also collected from two allografts of murine colorectal cancer (CT26) and B-cell lymphoma (A20) cells. Colorectal PDX tissue was subcutaneously implanted bilaterally into five male R2G2 and five male NSG mice. Growth was comparable between the R2G2 and the NSG mouse models; however, the standard error was much lower in the R2G2 strain. Head and neck PDX 626 and 635 was transplanted in 2.2 mm2 tissues into 4 sections of each of 2 R2G2 mice each (n=2/PDX), and 100% of mice developed either one or two tumors. The human esophageal adenocarcinoma OE33 cells were implanted into the left and right flanks of three each of R2G2, athymic nude and SCID mice. There was a 100% take rate in R2G2 mice, 0% in SCID mice and 17% in athymic nude mice. The human esophageal adenocarcinoma FLO1 cells were examined in two studies. In both studies, cells were injected into both flanks of R2G2 and SCID mice. Study A also examined growth in athymic nude mice. In study A, no tumor growth was seen in athymic nude or SCID mice, whereas the take rate was 100% in R2G2 mice. In study B, the take rate was 100% in both the R2G2 and the SCID mice, although differences were seen in growth rate. Human gastric adenocarcinoma AGS cells were implanted in both flanks of four each of R2G2 and SCID mice. The take rate was 75% in R2G2 mice and 0% in SCID mice. Head and neck squamous cell carcinoma SQ20b cells were implanted in twenty R2G2 mice and take rate was 90%. Growth of two allogeneic tumor lines was also examined. The mouse colon carcinoma CT26 cells were implanted in ten R2G2 mice and took in 100% of the mice. Mouse B-cell lymphoma A20 cells were implanted in ten R2G2 mice and take rate was 100%. Both allografts grew to 1000mm3 by 13 days post implantation. These data provide evidence that the R2G2 mouse model is a valuable tool for oncology programs including cell line tumor models research, with high take rates and quick growth of allogeneic models. Citation Format: Jamie L. Naden. Patient-derived and cell line xenograft growth in the B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1043.

Abstract 2033: Transforming growth factor-b-induced epithelial-mesenchymal transition is attenuated by telomerase-specific oncolytic virotherapy in human esophageal cancer

Abstract Background: Epithelial-mesenchymal transition (EMT) is known to be one of the poor prognostic factors. EMT is a biological process, by which epithelial cancer cells lose their epithelial characteristics and gain mesenchymal properties, including invasion, metastasis and drug resistance, resulting in tumor recurrence and poor prognosis. Esophageal cancer is one of the most common cause of cancer-related death worldwide, especially in East Asia. Despite the recent advances in the treatment of esophageal cancer, antitumor therapy to inhibit EMT program remains to be developed. We developed a telomerase-specific replication-selective oncolytic adenovirus (OBP-301; Telomelysin), in which the human telomerase reverse transcriptase promoter drives the expression of E1A and E1B for virus replication. In this study, we explored the biological effect of OBP-301 on EMT in human esophageal cancer cells. Methods: Transforming growth factor-β (TGF-β) was used to induce EMT in human esophageal cancer TE-4 cells. To investigate whether OBP-301 infection affects TGF-β-induced EMT, we performed western blot and real-time PCR analysis for EMT-related markers. We also examined the migration capability using transwell migration assay. In vitro antitumor effect of OBP-301 and chemotherapeutic agents on TGF-β-treated TE-4 cells were assessed by a XTT assay. Results: Administration of TGF-β induced mesenchymal characteristics, including spindle-shaped morphology, increased migration capability, and upregulation of mesenchymal markers N-cadherin and vimentin, in TE-4 cells. When TGF-β-treated TE-4 cells were infected with OBP-301, upregulation of N-cadherin and vimentin expression was attenuated. Upregulation of EMT-transcription factors, Snail, Slug, and ZEB1 by TGF-β was reduced by OBP-301 infection. OBP-301 also inhibited TGF-β-induced enhancement of migration capability. Moreover, interestingly, EMT-induced TE-4 cells acquired chemoresistance, whereas they were sensitive to OBP-301-mediated lytic effect. Conclusion: Our data suggest that telomerase-specific oncolytic virotherapy is a promising antitumor strategy to inhibit TGF-β-induced EMT and warrants clinical trials in human esophageal cancer. Currently, we are conducting a phase I/II clinical study of OBP-301 in combination with radiotherapy for esophageal cancer. Citation Format: Tomoya Masuda, Hiroshi Tazawa, Takeshi Ieda, Yuuri Hashimoto, Shunsuke Tanabe, Kazuhiro Noma, Yasuo Urata, Shunsuke Kagawa, Yasuhiro Shirakawa, Toshiyoshi Fujiwara. Transforming growth factor-b-induced epithelial-mesenchymal transition is attenuated by telomerase-specific oncolytic virotherapy in human esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2033.

PO-215 Patient-derived and cell line xenograft growth in the B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) mouse model

Introduction Immunodeficient mouse models are helping to advance the field of oncology. A new model on the market, the B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) knockout mouse, lacks responsiveness to common gamma chain cytokines, including IL-2, IL-4, IL-7, IL-9 and IL-15. In addition, this model exhibits defects in lymphoid development and so lacks mature lymphocytes of the B, T, and natural killer (NK) cell lineages. Herein we describe growth of multiple patient-derived (PDX) and tumour cell line xeno- (CDX) and allo-grafts in the R2G2 immunodeficient mouse model. Material and methods The PDXs examined include colorectal and head and neck cancers. The CDXs studied include esophageal (OE33 and FLO1) and stomach cancer (AGS). Tumour growth data was also collected from two allografts of murine colorectal cancer (CT26) and B-cell lymphoma (A20) cells. Results and discussions Colorectal PDX tissue was comparable between the R2G2 and the NSG mouse models, however the standard error was much lower in the R2G2 strain. Head and neck PDX 626 and 635 was transplanted in into 4 sections of R2G2 mice each, and 100% of mice developed either one or two tumours. The human esophageal adenocarcinoma OE33 cells were implanted into both flanks of R2G2, Athymic Nude and SCID mice. There was a 100% take rate in R2G2 mice, 0% in SCID mice and 17% in Athymic Nude mice. The human esophageal adenocarcinoma FLO1 cells were examined in two studies. In both studies, cells were injected into both flanks of R2G2 and SCID mice. Study A also examined growth in Athymic Nude mice. In study A, no tumour growth was seen in Athymic Nude or SCID mice, whereas the take rate was 100% in R2G2 mice. In study B, the take rate was 100% in both the R2G2 and the SCID mice, although differences were seen in growth rate. Human gastric adenocarcinoma AGS cells were implanted in both flanks of R2G2 and SCID mice. The take rate was 75% in R2G2 mice and 0% in SCID mice. Head and neck squamous cell carcinoma SQ20b cells were implanted in R2G2 mice and take rate was 90%. Growth of two allogeneic tumour lines was also examined. The mouse colon carcinoma CT26 cells were implanted in R2G2 mice and took in 100% of the mice. Mouse B-cell lymphoma A20 cells were implanted in R2G2 mice and take rate was 100%. Both allografts grew to 1000mm3 by 13 days post implantation. Conclusion These data provide evidence that the R2G2 mouse model is a valuable tool for oncology programs including cell line tumour models research, with high take rates and quick growth of allogeneic models.

Overexpression cyclin-dependent kinase 2A in esophageal cancer inhibiting its proliferation

Objective The aim of this study was to evaluate the overexpression cyclin-dependent kinase 2A (CDKN2A) gene in human esophageal cancer cell TE-13 and its function for proliferation. Methods Human esophageal cancer cell TE-13 was transfected with PcDNA3.1_CDNK2A and PcDNA3.1_neo as experiment and negative control group. The real-time quantitative polymerase chain reaction (Real-time PCR) was used to assess the CDNK2 expression after transfection. MMT assay and Transwell assay were used to examine the cell proliferation and invasion ability. Results CDNK2A mRNA relative expression were 0.45±0.10, 0.48±0.12 and 2.12±0.24 respectively with statistical difference in experiment group, negative group and blank control group respectively, which indicating CDNK2A mRNA can be overexpressed by transfecting PcDNA3.1_CDNK2 (F=200.500, P=0.000); D490 value were 0.71±0.08, 1.05±0.09 and 0.97±0.08 respectively with statistical difference (F=27.220, P=0.000). Transwell results showed the pass through biomembrane cells were (156.8±23.1), (166.9±28.4) and (162.1±30.3) cells without statistical difference (F=0.200, P=0.818). Conclusion Overexpression CDNK2A can inhibit human esophageal cancer cell TE-13 proliferation but not affect its invasion ability. Key words: Esophageal cancer; Cyclin-dependent kinase 2A gene; Proliferation; Invasion

Eupatilin inhibits the proliferation of human esophageal cancer TE1 cells by targeting the Akt‑GSK3β and MAPK/ERK signaling cascades.

Eupatilin, a type of flavonoid compound, has potential anti‑inflammatory and antitumor roles in gastric cancer and endometrial cancer; however, the effect of eupatilin on human esophageal cancer and the underlying molecular mechanisms remain unclear. In the present study, we investigated the antitumor effect of eupatilin on human esophageal cancer cells invitro and invivo. We found that eupatilin inhibited the proliferation and colony formation of esophageal cancer TE1 cells. DNA content analysis showed that eupatilin induced cell cycle arrest of TE1 cells at the G0/G1 phase. In addition, our results suggested that eupatilin suppressed TE1 cell proliferation by targeting the Akt/GSK3β and MAPK/ERK signaling cascades. Furthermore, treatment with eupatilin was found to decrease tumor volume in a TE1 xenograft mouse model, and the phosphorylation of Akt and ERK1/2 was inhibited by eupatilin in the tumor tissue. Notably, no obvious weight loss for the mice was detected. In conclusion, the present results indicate that the antiproliferative effect of eupatilin on esophageal cancer TE1 cells is associated with inhibition of the Akt and ERK pathways.

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.

Patient‐derived and cell line xenograft growth in the B6;129‐Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) mouse model

Immunodeficient mouse models are helping to advance the field of oncology. A new model on the market, the B6;129‐Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) knockout mouse, lacks responsiveness to common gamma chain cytokines, including IL‐2, IL‐4, IL‐7, IL‐9 and IL‐15. In addition, this model exhibits defects in lymphoid development and so lacks mature lymphocytes of the B, T, and natural killer (NK) cell lineages. Herein we describe growth of multiple patient‐derived (PDX) and tumor cell line xeno‐ (CDX) and allo‐grafts in the R2G2 immunodeficient mouse model. The PDXs examined include colorectal and head and neck cancers. The CDXs studied include esophageal (OE33 and FLO1) and stomach cancer (AGS). Tumor growth data was also collected from two allografts of murine colorectal cancer (CT26) and B‐cell lymphoma (A20) cells. Colorectal PDX tissue was subcutaneously implanted bilaterally into five male R2G2 and five male NSG mice. Growth was comparable between the R2G2 and the NSG mouse models, however the standard error was much lower in the R2G2 strain. Head and neck PDX 626 and 635 was transplanted in 2.2 mm2 tissues into 4 sections of each of 2 R2G2 mice each (n=2/PDX), and 100% of mice developed either one or two tumors. The human esophageal adenocarcinoma OE33 cells were implanted into the left and right flanks of three each of R2G2, Athymic Nude and SCID mice. There was a 100% take rate in R2G2 mice, 0% in SCID mice and 17% in Athymic Nude mice. The human esophageal adenocarcinoma FLO1 cells were examined in two studies. In both studies, cells were injected into both flanks of R2G2 and SCID mice. Study A also examined growth in Athymic Nude mice. In study A, no tumor growth was seen in Athymic Nude or SCID mice, whereas the take rate was 100% in R2G2 mice. In study B, the take rate was 100% in both the R2G2 and the SCID mice, although differences were seen in growth rate. Human gastric adenocarcinoma AGS cells were implanted in both flanks of four each of R2G2 and SCID mice. The take rate was 75% in R2G2 mice and 0% in SCID mice. Head and neck squamous cell carcinoma SQ20b cells were implanted in twenty R2G2 mice and take rate was 90%. Growth of two allogeneic tumor lines was also examined. The mouse colon carcinoma CT26 cells were implanted in ten R2G2 mice and took in 100% of the mice. Mouse B‐cell lymphoma A20 cells were implanted in ten R2G2 mice and take rate was 100%. Both allografts grew to 1000mm3 by 13 days post implantation. These data provide evidence that the R2G2 mouse model is a valuable tool for oncology programs including cell line tumor models research, with high take rates and quick growth of allogeneic models.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Simultaneous Multiparameter Cellular Energy Metabolism Profiling of Small Populations of Cells

Functional and genomic heterogeneity of individual cells are central players in a broad spectrum of normal and disease states. Our knowledge about the role of cellular heterogeneity in tissue and organism function remains limited due to analytical challenges one encounters when performing single cell studies in the context of cell-cell interactions. Information based on bulk samples represents ensemble averages over populations of cells, while data generated from isolated single cells do not account for intercellular interactions. We describe a new technology and demonstrate two important advantages over existing technologies: first, it enables multiparameter energy metabolism profiling of small cell populations (<100 cells)—a sample size that is at least an order of magnitude smaller than other, commercially available technologies; second, it can perform simultaneous real-time measurements of oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and mitochondrial membrane potential (MMP)—a capability not offered by any other commercially available technology. Our results revealed substantial diversity in response kinetics of the three analytes in dysplastic human epithelial esophageal cells and suggest the existence of varying cellular energy metabolism profiles and their kinetics among small populations of cells. The technology represents a powerful analytical tool for multiparameter studies of cellular function.

The G2 phase arrest induced by sterigmatocystin is dependent on hMLH1- ERK/p38-p53 pathway in human esophageal epithelium cells in vitro

Sterigmatocystin (ST), being a precursor of aflatoxin, is categorized as Group 2B carcinogen. Our previous studies found that both mismatch repair (MMR) pathways and p53 signaling pathway were involved in ST-induced G2 cell cycle arrest in human esophageal squamous epithelial cell line, HET-1A, in vitro. Studies showed that ERK, JNK and p38 signaling pathways played important roles in cell cycle arrest induced by several other carcinogens. However, the role of MAPK pathway and the links between the MMR and p53 signaling pathways in ST induced G2 phase arrest is still not clarified. In the present study, we first explored the role of MAPK pathway upon ST induced G2 arrest, and found that ST up-regulated the expression of G2/M regulatory factors through MAPK signaling pathway (both ERK and p38, but not JNK pathway). The inhibition of ERK and p38 significantly inhibited p53 activation by ST. Blockage of MMR pathway by silencing hMLH1 expression inhibited ERK, p38 and p53 activation and then attenuated G2 arrest by ST. Thus, in conclusion, the current study demonstrated that in response to ST induced DNA damage, hMLH1 was first activated, then triggered ERK, p38 and p53 activation and finally resulted in G2 arrest in HET-1A cells.

Helicobacter pylori induces caudal-type homeobox protein 2 and cyclooxygenase 2 expression by modulating microRNAs in esophageal epithelial cells.

Dysregulation of microRNAs (miRNAs) has been linked to virulence factors of Helicobacter pylori. The role of H. pylori in esophageal disease has not been clearly defined. We previously reported that H. pylori esophageal colonization promotes the incidence of Barrett's esophagus and esophageal adenocarcinoma in vivo. Here, we studied the direct effects of H. pylori on the transformation of esophageal epithelial cells, with particular focus on whether H. pylori exerts its effects by modulating miRNAs and their downstream target genes. The normal human esophageal cell line HET‐1A was chronically exposed to H. pylori extract and/or acidified deoxycholic acid for up to 36 weeks. The miRNA profiles of the esophageal epithelial cells associated with H. pylori infection were determined by microarray analysis. We found that chronic H. pylori exposure promoted acidified deoxycholic acid‐induced morphological changes in HET‐1A cells, along with aberrant overexpression of intestinal metaplasia markers and tumorigenic factors, including caudal‐type homeobox protein 2 (CDX2), mucin 2, and cyclooxygenase 2 (COX2). Helicobacter pylori modified the miRNA profiles of esophageal epithelial cells, particularly aberrant silencing of miR‐212‐3p and miR‐361‐3p. Moreover, in biopsies from Barrett's esophagus patients, esophageal H. pylori colonization was associated with a significant decrease in miR‐212‐3p and miR‐361‐3p expression. Furthermore, we identified COX2 as a target of miR‐212‐3p, and CDX2 as a target of miR‐361‐3p. Helicobacter pylori infection of esophageal epithelial cells was associated with miRNA‐mediated upregulation of oncoprotein CDX2 and COX2. Our observations provide new evidence about the molecular mechanisms underlying the association between H. pylori infection and esophageal carcinogenesis.

Gecko Active Components Regulates Endoplasmic Reticulum Stress to Induce the Apoptosis of KYSE150 Cells through PERK Pathway

Objective: Gecko active components (GACs), extracted from the powder of whole Gecko, have been reported to be effective against esophageal squamous cell carcinoma (ESCC). Endoplasmic reticulum stress (ERs) has been regarded as an important cause for pathogenesis of esophageal squamous cell carcinoma (ESCC). In this paper, we aimed to study the effect of GACS on apoptosis of human esophageal carcinoma KYSE150 cells and to analyze the underlying signaling pathway. Methods: MTT assay was used to detect the viability of KYSE150 cells, and Flow cytometry was applied to detect reactive oxygen species (ROS), calcium generation, and the level of mitochondrial membrane potential (MMP). Western blot analysis was applied to observe the expression of apoptosis-related proteins and endoplasmic reticulum stress (ERs)-related proteins in KYSE150 cells. Results: The results showed that GACs inhibited KYSE150 cell vitality in a dose- and time-dependent manner. Not only that, GACs could up-regulated ERs-related and apoptosis-related proteins expression, and the content of ROS and calcium were significantly increased, and also, the level of MMP was significantly decreased. Conclusion: The results of this report suggested that induction of apoptosis occurs through the ERs dependent signaling pathways.

The role of NRAGE subcellular location and epithelial-mesenchymal transition on radiation resistance of esophageal carcinoma cell.

Neurotrophin receptor-interacting MAGE homolog (NRAGE) has been considered as a tumor suppressor. In the previous study, we established human esophageal carcinoma resistance cell line TE13R120 and found the difference of NRAGE expression between TE13 and TE13R120 cells by gene microarray. Herein, we further discuss the possible molecular mechanism of NRAGE on participating the radiation sensitivity of esophageal carcinoma cells. We used colony formation assay to measure the surviving fraction and relevant radiobiological parameters. NRAGE expression was estimated by immunofluorescence and Western blot. Tumor growth factor-β (TGF-β) was used for inducing epithelial-mesenchymal transition (EMT) in TE13 cells to detect the relationship between NRAGE and EMT; the capacity of cell migration was also assessed by wound healing assay. TE13R120 cells were showed significantly radioresistance compared with TE13. The D0, Dq, and N value of TE13R120 were all higher than those of TE13 (2.499, 1.991, and 2.219 vs. 2.242, 0.854, and 1.645), as well as SF2 (0.734 vs. 0.538). Results of immunofluorescences showed that NRAGE was mainly expressed in the nucleus of TE13R120 cells, but in TE13 cells, it was mainly in cytoplasm. In addition, EMT phenotype was observed in TE13R120 cells and TGF-β-induced EMT in TE13 cells, E-cadherin expression was decreased, but vimentin was upregulated. Furthermore, TE13 cells have a rising tendency in NRAGE nucleus expression after treatment with TGF-β. Results of wound healing assay showed that the cell migration of TE13R120 and TGF-β-induced EMT in TE13 cells were remarkably enhanced. Our results indicate that NRAGE subcellular localization is related to radiation resistance of esophageal carcinoma cell and EMT may be involved in NRAGE subcellular location.

Experimental study on the contribution of costimulatory molecule B7-H3 in regulation of the biological function of human esophageal cancer cell line Eca-109

Objective To investigate the role of B7-H3 in the regulation of biological function of human esophageal cancer cell by using RNA interference (RNAi) methods. Methods Obtaining LV-B7-H3-siRNA and negative control LV-NC-Eca-109 by lentivirus infecting and followed by fluorescence-activated cell sorter (FACS) Aria II cell sorting system. The expression level of B7-H3 in LV-B7-H3-siRNA-Eca-109 cells and LV-NC-Eca-109 was confirmed by using real-time quantitative polymerase chain reactionB (Real-time PCR), FACS and Western blotting methods. The cell proliferation was determined by cell counting kit-8 (CCK-8) assay, and the migration and invasion was determined by wound healing assay as well as transwell assay respectively. Results We constructed the recombinant lentivirus of siRNA targeting B7-H3. On day 2 (0.31±0.03 vs. 0.36±0.02), 4 (0.51±0.04 vs. 0.60±0.04), 5 (0.60±0.02 vs. 0.70±0.03) and day 6 (0.65±0.05 vs. 0.76±0.03), the proliferation rate of LV-B7-H3-siRNA group was significantly lower than that of LV-NC control group by using CCK-8 assay (P=0.011, 0.024), respectively. The wound healing assay on Eca-109 cells in LV-B7-H3-siRNA group as well as LV-NC group showed that, the cell-free area of the LV-B7-H3-siRNA group was significantly wider than that of LV-NC group at 12 h [(38.0±28.1) vs. (467.7±30.3) pix, P=0.042], 20 h [(509.7±6.2) vs. (403.3±16.7) pix, P=0.001] and 24 h [(454.7±19.9) vs. (355.0±32.1) pix, P=0.010] respectively after drawing the scratch line on the monolayer cells. The transwell invasion assay showed that at the time point of 24 h, the number of invaded cells in the LV-B7-H3-siRNA group (258.8±29.1) cells was significantly lower than that in the LV-NC group (474.0±85.3) cells (P=0.014). Conclusion We successfully constructed the recombinant lentivirus of siRNA targeting B7-H3, and the cellular studies showed that the down-regulation of B7-H3 could suppress the biological functions such as proliferation, migration and invasion of the Eca-109 cells. Thus the over-expression of B7-H3 may involve in the malignant transformation of human esophageal cancer. The B7-H3 molecule could be developed as the potential therapeutic targets for immunotherapy of this malignancy. Key words: Co-stimulatory molecule; B7-H3; Esophageal cancer; Invasion; Metastasis

RhoE expression is negatively correlated with epidermal growth factor receptor and its prognosis value in human esophageal squamous cell carcinoma

Objective To investigate the gene expression of RhoE, a small Rho GTP protein in esophageal squamous cell carcinoma (ESCC) tissues and its association with epidermal growth factor receptor (EGFR) expression and patients prognosis. Methods Thirty patietns with confirmed diagnosis of ESCC were included in this study form May 10 2015 to July 10 2016. Real time quantitative PCR was applied to detect the expression of RhoE and EGFR in paired ESCC and corresponding normal tissues. The prognosis of RhoE was further evaluated based on the recorded survival time of the 30 ESCC patients. Results Comparing with the normal esophageal tissues, RhoE was significantly downregulated in ESCC. 63.3% of these ESCC tissues expressed more than 2 fold downregulation of RhoE. Besides, EGFR gene was upregulated in these corresponding ESCC tissues. A significant negative correlation was found between RhoE and EGFR expression (r=-0.550, P=0.032). Kaplan-Meier analysis of association between RhoE expression and overall survival of these ESCC patients revealed that lower RhoE levels indicated poorer prognosis [hazard ratio (HR)=0.210, P=0.038]. Conclusion Down-regulation of RhoE was observed in ESCC tissues, which negatively correlated with EGFR expression and prognosis. All these indicate the vital tumor suppressing roles of RhoE in the development of ESCC. Key words: RhoE; Epidermal growth factor receptor; Esophageal squamous cell carcinoma; Prognosis

Selective anticancer activity of the novel steroidal dihydropyridine spirooxindoles against human esophageal EC109 cells

A series of small-molecule compounds built on steroidal dihydropyridine spirooxindoles has been reported previously. In this study, the compound 5l showed strong anti-cancer activity, especially in the esophageal cancer. Three esophageal squamous cell lines and paclitaxel-resistant cell line were investigated. The results demonstrated that compound 5l was most efficient in the EC109 cells, induced cell apoptosis through elevation of cellular ROS levels, caused G2/M phase arrest and mitochondrial dysfunction. Further study confirmed that the mechanism of 5l in esophageal cancer treatment was related to the Bcl-2 family and caspase receptor-mediated apoptotic pathway.

MicroRNA-615-5p targets insulin-like growth factor 2 and exerts tumor-suppressing functions in human esophageal squamous cell carcinoma

To investigate the expression pattern, clinical significance and functional roles of microRNA (miR)-615-5p in human esophageal squamous cell carcinoma(ESCC), , quantitative real-time PCR was performed to detect expression levels of miR‑615‑5p in ESCC tissues and cell lines. Associations between miR‑615‑5p expression and various clinicopathological features of ESCC patients were also statistically evaluated. The candidate targets of miR‑615‑5p were identified by integrating bioinformatics miRNA target prediction, western blot analysis and luciferase reporter assay. Moreover, the functions of miR‑615‑5p in ESCC cell migration and invasion were determined using the transfection of miRNA mimics, or co-transfected with miRNA mimics and the expression vector of its target gene. As a result, miR‑615‑5p expression in ESCC tissues and cells were markedly lower than those in non-cancerous esophageal mucosa and human normal esophageal cells, respectively (both P<0.001). miR‑615‑5p downregulation was significantly associated with advanced tumor-node-metastasis stage, positive lymph node metastasis and moderate-poor differentiation. Functionally, the re-expression of miR‑615‑5p suppressed the invasion and migration of ESCC cells invitro. Interestingly, insulin-like growth factor 2 (IGF2) was identified as a direct target gene of miR‑615‑5p, and the inhibitory effects of miR‑615‑5p in ESCC cell motility were reversed by the restoration of IGF2 expression. In conclusion, miR‑615‑5p downregulation may be an underlying molecular mechanism of development and progression of ESCC, and may function as a potential therapeutic target of this malignancy. Also, we illustrate that the miR‑615‑5p/IGF2 axis may bring important contributions to cell motility of human ESCC.