Human Esophageal Squamous Cell Research Articles

COX-2 strengthens the effects of acid and bile salts on human esophageal cells and Barrett esophageal cells

AimsInvestigate the effect and mechanism of COX-2 on viability, intestinal metaplasia, and atypia in human esophageal squamous and Barrett esophageal cell lines.MethodsHuman esophageal squamous and Barrett esophageal cell lines were transfected with a COX-2 expression vector and a COX-2 siRNA, and then were treated with acid, bile salts, and a mixture of both. Cell viability, the expression of COX-2, NF-κB(p65), CDX-2, MUC2, c-myb, and BMP-4, and the morphology and microstructure of cells were then observed.ResultsThe viability of COX-2 overexpressed cells was significantly higher than that of control cells, while the viability of COX-2 siRNA-treated cells was significantly lower than that of control cells. Intestinal metaplasia and atypia were observed in cells overexpressing COX-2. Acid, bile salts, and their mixture inhibited the viability of these two cell lines, but the inhibitory effect of the mixture was stronger than a single treatment in either. SiRNA mediated knockdown of COX-2 strengthened the antiproliferative effects of the mixture on HET-1A and BAR-T cells. The expression of p-p65, CDX-2, and BMP-4 was positively correlated with COX-2 expression, while the expression levels of p65, MUC2, and c-myb remained unchanged.ConclusionCOX-2 may influence the viability, atypia, and intestinal metaplasia of human esophageal cells and Barrett esophageal cells. Activation of the p-p65, CDX-2, and BMP-4 signaling pathways by COX-2 may be part of this mechanism.

Carvacrol Induces Cell Apoptosis in Human Esophageal Squamous Carcinoma the Human Esophageal Squamous Carcinoma Cell Line (KYSE-150) Cells

Carvacrol is a natural phenol with antioxidant, antimicrobials, and anti-cancer activities. The present study aimed to explore the anti-cancer activity of carvacrol in human esophageal squamous carcinoma KYSE-150 cells. Cell proliferation and apoptosis were measured using WST-1 assay and cell death detection ELISA kit respectively. Caspase activities were determined with caspase fluorometric assay kits. WST-1 assay revealed that carvacrol suppressed KYSE-150 cell proliferation in both dose and time-dependent manner. Carvacrol could increase cell apoptosis in a dose-dependent manner through caspase-3 and caspase-9 activation. In addition, Bcl-2 mRNA levels were significantly decreased by carvacrol treatment. This study delivers that carvacrol exerts a favorable anti-cancer action against esophageal cancer via inhibiting cell proliferation and inducing cell apoptosis. These discoveries suggest that carvacrol is deserved to be further studied and developed as a chemotherapeutic agent for esophageal cancer.

Gastroesophageal Junction Fat Pad Tissue from Obese Patients Impairs Esophageal Epithelial Barrier Function and Disrupts Cell‐to‐Cell Connections

Introduction Visceral obesity is associated with GERD and, traditionally, it has been assumed that visceral fat predisposes to GERD primarily by increasing intra-abdominal pressure. However, transmission electron microscopy (TEM) of esophageal biopsies from obese individuals without GERD exhibit dilated intercellular spaces (DIS) and, in earlier studies, we found that substances secreted by mature adipocytes in visceral fat from obese patients impair esophageal epithelial barrier function, induce DIS and reduce transepithelial resistance (TER). There is a fat pad at the gastroesophageal junction (GEJ) that is in intimate contact with the distal esophagus. We now explore whether whole GEJ fat tissue of obese individuals secretes substances that impair esophageal barrier function. Methods We obtained GEJ fat from 3 obese (BMI 46, 60, 74) and 3 non-obese (BMI 24, 26, 27) patients having foregut surgery. Human esophageal squamous cells were seeded into transwells and grown to confluence for 3 days. On day 4, cells were raised to an air-liquid interface (ALI) to allow for differentiation, stratification, and esophageal barrier formation. ALI cultures were exposed to conditioned media (CM) from GEJ fat or to control medium beginning at day 4. TER was measured on days 4, 6, and 8 using the EVOM2 Epithelial Volt/Ohm Meter. On day 8, ALI cultures were stained with H&E and Ki-67 and imaged; barrier permeability was assessed by a biotinylation reagent applied to the ALI surface. TEM of ALIs and of esophageal biopsies from 2 non-obese and 2 obese patients without GERD were assessed. Results CM from all 3 obese patients significantly reduced TER on days 6 and 8; CM from 2 non-obese patients did not reduce TER whereas CM from 1 non-obese patient induced a slight reduction in TER at day 8 (Table 1). Marked DIS were found only in ALI cultures grown with CM from obese patients. In ALIs grown with CM from non-obese patients, the biotinylation reagent stopped at the superficial layers of esophageal cells. In contrast, the biotinylation reagent diffused through all cell layers in ALIs grown with obese patient CM. Increases in basaloid cells and in Ki-67 were found in ALIs grown with obese patient CM. TEMs of ALIs grown with non-obese patient CM demonstrated well-oriented cell-cell junctions similar to TEMs of non-obese patient esophageal biopsies. In contrast, ALIs grown with obese patient CM demonstrated cell-cell junctional structures that were disorganized, aggregated, and floating within the DIS, similar in appearance to TEMs of the obese patients’ esophageal biopsies. Conclusion Substances produced by GEJ fat of obese patients impair esophageal epithelial barrier integrity, cause basal cell hyperplasia and DIS, and disrupt intercellular connections. These findings in ALI cultures recapitulate abnormalities seen by TEM in esophageal biopsies of obese patients.

GATA4 blocks squamous epithelial cell gene expression in human esophageal squamous cells

GATA4 promotes columnar epithelial cell fate during gastric development. When ectopically expressed in the developing mouse forestomach, the tissue emerges as columnar-like rather than stratified squamous with gene expression changes that parallel those observed in the pre-malignant squamous to columnar metaplasia known as Barrett’s esophagus (BE). GATA4 mRNA up-regulation and gene amplification occur in BE and its associated cancer, esophageal adenocarcinoma (EAC), and GATA4 gene amplification correlates with poor patient outcomes. Here, we explored the effect of ectopic expression of GATA4 in mature human esophageal squamous epithelial cells. We found that GATA4 expression in esophageal squamous epithelial cells compromised squamous cell marker gene expression and up-regulated expression of the canonical columnar cell cytokeratin KRT8. We observed GATA4 occupancy in the p63, KRT5, and KRT15 promoters, suggesting that GATA4 directly represses expression of squamous epithelial cell marker genes. Finally, we verified GATA4 protein expression in BE and EAC and found that exposure of esophageal squamous epithelial cells to acid and bile, known BE risk factors, induced GATA4 mRNA expression. We conclude that GATA4 suppresses expression of genes marking the stratified squamous epithelial cell lineage and that this repressive action by GATA4 may have implications in BE and EAC.

Synthesis and in vitro antiproliferative activities of lupanol derivatives towards human esophageal squamous carcinoma cells

A series of lupanol derivatives were synthesized and evaluated in vitro for their inhibitory activities against three human esophageal squamous carcinoma cells lines, Eca-109, TE-1 and EC-9706. Among lupanol derivatives, seven were new compounds, and lupanol cinnamate analogues 5 and 6, hydrazone analogues 9 and 10 presented high activities towards all the tested tumour cells, even higher activities than those of doxorubicine.

Products of Mature Adipocytes in Visceral Fat of Obese Patients Impair Esophageal Epithelial Barrier Formation and Function

ObjectiveVisceral obesity is associated with GERD and its complications of reflux esophagitis, Barrett’s esophagus and esophageal adenocarcinoma. A traditional explanation for these associations is that visceral fat causes pathological acid reflux by increasing intra‐abdominal pressure. However, for unknown reasons, the esophagus of obese patients who have normal esophageal acid exposure can exhibit evidence of impaired epithelial barrier function including dilated intercellular spaces (DIS) and reduced transepithelial resistance (TER). In this study, we explored our hypothesis that the visceral fat of obese individuals secretes substances that impair esophageal barrier function, thus predisposing to injury by GERD.MethodsWe obtained subcutaneous (SQ) and visceral (VIS) fat from 1 non‐obese (BMI 18) and 2 obese (BMIs 51& 42) patients having foregut surgery; VIS included both mesenteric fat and gastroesophageal junction (GEJ) fat pad. Fat contains both mature adipocytes (MAs) and adipocyte derived stem cells (ADSCs), which were isolated from adipocyte tissues by collagenase digestion and differential centrifugation; immunofluorescence (CD90+, CD44+, CD31−, CD45−) was used to confirm ADSC isolation. Telomerase‐immortalized normal, human esophageal squamous cells (NES‐B10T) were seeded into transwells and grown to confluence for 3 days. On day 4, the cells were raised to an air‐liquid interface (ALI) to allow for differentiation, stratification, and formation of an esophageal epithelial barrier. ALI cultures were exposed to conditioned media (CM) from MAs or ADSCs, or to control NES medium beginning at day 4. TER was measured (in Ω·cm2) on days 4, 6, and 8 using the EVOM2 Epithelial Volt/Ohm Meter (World Precision Instruments, Sarasota, FL). On day 10, ALI cultures were stained with H&E and imaged.ResultsCM from ADSCs of SQ and VIS fat from an obese patient did not reduce the TER of NES ALI cultures at any time point; CM from MAs of SQ fat from all 3 patients also did not reduce TER. In contrast, CM from MAs of VIS fat from both obese patients significantly reduced TER on day 6 compared with control medium; TER also remained lower than control on day 8 (Table 1). CM from MAs of VIS fat from the non‐obese patient did not reduce TER of NES ALI cultures at any time point (Table 1). H&E staining demonstrated DIS only in those ALI cultures grown with the CM from MAs of VIS fat from the obese patients.ConclusionsSubstances produced by MAs, but not by ADSCs, in the VIS fat of obese patients impair esophageal epithelial barrier integrity and cause morphologic changes of DIS. No such effects were found for substances produced by MAs in the SQ fat of any patient, or from those produced by MAs in VIS fat of the non‐obese patient. Moreover, VIS fat in the mesentery and at the GEJ exert similar functional and morphologic effects on esophageal barrier formation. We are conducting further investigations to identify the mechanisms underlying the impairment of esophageal barrier formation caused by visceral adiposity, specifically for the GEJ fat pad that surrounds the distal esophagus.Support or Funding InformationBaylor Scott & White Research Institute Transepithelial Resistance (Ω·cm2 ±SEM) of NES on Day 6 and Day 8 of ALI Culture Exposed to Conditioned Media from Mature Adipocytes from Subcutaneous (SQ), Mesenteric, and GEJ Adipocyte Tissues BMI Control Day 6 SQ Day 6 Mesenteric Day 6 GEJ Day 6 Control Day 8 SQ Day 8 Mesenteric Day 8 GEJ Day 8 18 241±2.1 242±2.0 234±2.0 235±2.5 245±3.8 256±2.2 243±4.2 248±7.2 51 212±2.0 204±3.5 194±3.0 * 192±0.5 ** 235±8.5 233±1.0 209±6.5 207±8.0 42 264±3.6 263±5.5 236±2.3 ** 239±3.3 ** 251±2.4 296±45 249±0.9 237±2.6 * *p≤0.05; **p≤0.01 compared with control

Epigenetic silencing of IGFBPL1 promotes esophageal cancer growth by activating PI3K-AKT signaling

BackgroundThere are seven insulin-like growth factor binding proteins (IGFBPs) that bind insulin-like growth factors (IGFs). IGFBP like protein1 (IGFBPL1) is a new member of this family. The function and mechanism of IGFBPL1 in esophageal cancer remains to be elucidated.MethodsEight esophageal cancer cell lines, 114 cases of esophageal dysplasia, and 501 cases of primary esophageal cancer samples were examined in this study. Methylation-specific polymerase chain reaction (MSP), immunohistochemistry, Western blot, flow cytometry, RNA interference assay, and xenograft mouse models were employed.ResultsThe expression of IGFBPL1was lost and complete methylation was found in KYSE150 and KYSE410 cells. Reduced expression and partial methylation of IGFBPL1 was found in Bic1, KYSE140, KYSE450, KYSE520, and COLO680N cells. High expression and unmethylation was detected in KYSE510 cells. Restoration of IGFBPL1 expression was found in KYSE150 and KYSE410 cells and the expression of IGFBPL1 was increased in Bic1, KYSE140, KYSE450, KYSE520, and COLO680N cells, after 5-AZA-2′-deoxycytidine treatment. IGFBPL1 was methylated in 47.3% (53/114) of esophageal dysplasia and 49.1% (246/501) of human primary esophageal squamous cell carcinoma (ESCC). Methylation of IGFBPL1 was significantly associated with TNM stage (p = 0.012), and tumor size (p = 0.009). IGFBPL1 inhibited esophageal cancer cell clonal formation and proliferation and induced cell apoptosis and G1/S phase arrest. Further study found that IGFBPL1 is involved in PI3K-AKT signaling and IGFBPL1 suppressed human ESCC xenografts growth in mice.ConclusionIGFBPL1 suppresses esophageal cancer cell growth by inhibiting PI3K-AKT signaling in vitro and in vivo. IGFBPL1 is a novel tumor suppressor in human esophageal cancer.

Licochalcone C induces cell cycle G1 arrest and apoptosis in human esophageal squamous carcinoma cells by activation of the ROS/MAPK signaling pathway

Along with changes in dietary habits and lifestyle, the incidence of esophageal cancer is increasing around the world. Since chemotherapy for esophageal cancer has significant side effects, phytochemicals have attracted attention as an alternative medicine. Licochalcone C (LCC) is a flavonoid compound extracted from Licorice, with a variety of clinical uses including anti-cancer, anti-inflammatory and anti-oxidant effects. Treatment with LCC for 48 h significantly decreased cell viability of esophageal squamous cell carcinoma (ESCC) cells in a dose- and time-dependent manner with IC50 values of 28 µM (KYSE 30), 36 µM (KYSE 70), 19 µM (KYSE 410), 28 µM (KYSE 450) and 26 µM (KYSE 510). LCC induced G1 arrest accompanied by decreased cyclin D1 expression and an increase in the levels of p21 and p27. LCC increased the levels of intracellular ROS, cytochrome C release, and multi-caspase activity, and decreased mitochondrial membrane potential. LCC induced the protein expression of ER stress markers (GRP78 and CHOP) and phosphorylation JNK, c-Jun and p38. We investigated the expression of pro-apoptotic and anti-apoptotic proteins to elucidate the mechanism of apoptosis. Our findings contribute to the understanding of apoptosis mechanism underlying LCC in ESCC cells and provide new insights into the potential clinical opportunities of LCC for ESCC treatment.

MiR-760靶向c-Myc对人食管鳞状细胞癌TE-10细胞生物学特性的影响

miR-760靶向c-Myc对人食管鳞状细胞癌TE-10细胞生物学特性的影响

Effect of HMGB1 knockdown by RNA interference on cell proliferation and migration of esophageal squamous cell carcinoma after X-ray radiation

Objective To evaluate the effect of X-ray radiation on cell proliferation, migration, survival ability and cell cycle of human esophageal squamous cell carcinoma after RNA interference-mediated down-regulation of HMGB1 gene expression. Methods The expression of HMGB1 at mRNA and protein levels in the human esophageal squamous cell carcinoma cell lines ECA109 and KYSE30 was determined using RT-PCR and Western blot assays. MTS and Transwell assays were employed to examine the proliferation and migration of ECA109 and KYSE30 cell lines. The cellular survival ability in vitro was assessed by clone formation assay. The cell cycle after X-ray radiation in different groups was detected by flow cytometry. Results The expression of HMGB1 at mRNA and protein levels in ECA109 and KYSE30 cells were markedly higher in a dose-dependent and time-dependent manner in the radiation group than that in the control group (all P<0.05). MTS results demonstrated that the proliferation of ECA109 and KYSE30 cells was obviously lower at each time point after radiation than that in the group without radiation (all P<0.01). The expression of HMGB1 at mRNA and protein levels was significantly inhibited in the HMGB1 siRNA group than those in the control and NC groups (both P<0.01). The data from the clone formation assay revealed that the radiosensitivity was significantly increased after down-regulation of HMGB1 expression (P<0.01). Transwell migration assay revealed that the number of migrating cells at the fourth hour after X-ray irradiation in the HMGB1 siRNA group was significantly lower than those in the control and negative groups (both P<0.01). In the HMGB1 siRNA group, the percentage of cells at G0/G1 phase was obviously higher, whereas the percentage of S phase was significantly lower than those in the control and NC groups, and the trend was even more significant after X-ray radiation (all P<0.01). Conclusion Inhibition of HMGB1 expression by siRNA can suppress the proliferation and migration of ECA109 and KYSE30 cells and enhance the radiosensitivity by increasing the cell cycle arrest at G0/G1 stage after X-ray irradiation in vitro. Key words: HMGB1 gene; Cell proliferation and migration; Cell cycle; Radiosensitivity

Glutathione S-transferase omega 2 regulates cell growth and the expression of E-cadherin via post-transcriptional down-regulation of β-catenin in human esophageal squamous cells.

Glutathione S-transferase omega 2 (GSTO2), which belongs to the superfamily of GST omega class, lacks any appreciable GST activity. Although GSTO2 exhibits thioltransferase and glutathione dehydrogenase activities, its precise expression and physiological functions are still unclear. In the present study, we found that GSTO2 is exclusively expressed in the basal cell layer in Ki67-negative non-proliferative cells in the human esophageal mucosa. GSTO2 overexpression in esophageal squamous cell carcinoma (ESCC) cell lines inhibited cell growth and colony formation, and GSTO2-transfected cells formed smaller tumors in nude mice compared with mock-transfected cells. Interestingly, GSTO2 induction suppressed the expressions of E-cadherin and β-catenin at the cell-cell contact site. We quantified the phosphorylation levels of key proteins of MAPK signaling pathway and identified phosphorylation of p38. Additionally, HSP27, a downstream molecule of p38, was accelerated in GSTO2-transfected cells, unlike in mock-transfected cells. When GSTO2-transfected cells were treated with a p38 inhibitor, the expression of β-catenin and the membrane localization of E-cadherin was recovered. We next examined GSTO2 expression in 61 ESCC tissues using quantitative reverse transcription polymerase chain reaction and immunostaining. The results showed that GSTO2 mRNA and protein were significantly reduced in ESCC compared with normal tissues. When human ESCC cell lines were treated with 5-aza-2'-deoxycytidine, a DNA-methyltransferase inhibitor, GSTO2 transcription was induced, suggesting that aberrant hypermethylation is the cause of the down-regulated expression. Our results indicate that GSTO2 expression inhibits the membrane localization of E-cadherin, probably by modulation of the p38 signaling pathway. Down-regulation of GSTO2 by DNA hypermethylation contributes to the growth and progression of ESCC.

Interleukin-23 receptor signaling mediates cancer dormancy and radioresistance in human esophageal squamous carcinoma cells via the Wnt/Notch pathway

In the tumor microenvironment, inflammatory cells and molecules influence almost every process; among them, interleukin-23 (IL-23) is a pro-inflammatory molecule that exhibits pro- or anti-tumor properties, but both activities remain poorly understood. In this study, we investigated the effect of extracellular IL-23 in IL-23 receptor-positive (IL-23R+) esophageal squamous cell carcinoma (ESCC) and explored the mechanisms underlying this effect. We analyzed ESCC tumor tissues by immunohistochemical and immunofluorescence staining and found that IL-23, which was highly expressed, co-localized with Oct-4A in IL-23R+ ESCC cells. In addition, IL-23 treatment significantly increased the accumulation of CD133+ cells and activated the Wnt and Notch signaling pathways in CD133−IL-23R+ ESCC cell lines. Consistently, CD133−IL-23R+ cells pretreated with IL-23 showed stronger anti-apoptosis activity when exposed to radiation and higher survival than untreated groups. Moreover, the inhibition of Wnt/Notch signaling by a small-molecule inhibitor or siRNA abolished the effect of IL-23-induced dormancy and consequent radioresistance. Taken together, these results suggested that IL-23 facilitates radioresistance in ESCC by activating Wnt/Notch-mediated G0/1 phase arrest, and attenuating these detrimental changes by blocking the formation of dormancy may prove to be an effective pretreatment for radiotherapy.Key messagesIL-23/IL-23R is correlated with the acquisition of stem-like potential in ESCC.CD133−IL-23R+ ESCCs acquired dormancy via IL-23.Radioresistance depends on IL-23-mediated Wnt/Notch pathway activation in vitro and vivo.

Expression of PSMD7 in human esophageal squamous cell carcinoma specimens and its effects on cell proliferation and apoptosis

Expression of PSMD7 in human esophageal squamous cell carcinoma specimens and its effects on cell proliferation and apoptosis

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

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

Expression of growth inhibitory factor 2 in human esophageal squamous cell carcinoma and clinical significance

Objective To investigate the expression of growth inhibitory factor 2 (ING2) in esophageal squamous cell carcinoma (ESCC), and explore its correlation with lymphatic metastasis and prognosis. Methods The expression of ING2 in 63 cases of ESCC tissues and 63 cases of normal tissues was detected by immunohistochemistry. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to examine the expression of ING2 mRNA in fresh tumoral and normal tissues. Results The ING2 positive staining was predominantly localized in both nucleus and cytoplasm of cancer cells, but weakly in nucleus of normal epithelial cells only. The expression of ING2 protein and mRNA was significantly higher in ESCC tissues than in normal tissues (P=0.001). Moreover, the expression level of ING2 protein in ESCC tissues was significantly correlated with grade (P=0.010) and lymph node metastasis (P=0.001). Cox regression analysis revealed that ING2 expression level (P=0.042) and lymph node metastasis (P=0.000) were independent prognostic factors of ESCC. Conclusion ING2 is overexpressed in ESCC tissues. High expression of ING2 correlates with pathological grades, lymphatic metastasis and unfavorable survival in ESCC. Key words: Esophageal neoplasms; Squamous cell; Growth inhibitory factor 2; Metastasis; Prognosis

Abstract 2694: Esophageal cancer exhibits a resistance to the chemical inhibition of IGF-1R with a maintained Ras-MAPK activity

Abstract The type 1 insulin-like growth factor receptor (IGF-1R) and its associated signaling system play a significant role in carcinogenesis and progression of gastrointestinal malignancies, and thus have provoked great interest as a promising target for cancer treatment. The aim of our study was to assess the effects of a novel IGF-1R inhibitor, NVP-AEW541, on the cell proliferation and signal transduction of esophageal squamous cell carcinoma. Five human esophageal squamous carcinoma cell lines (TE-1, TE-4, TE-8, TE-10 and T.Tn) were treated with this inhibitor and the IC50 of NVP-AEW541 for each cell line was more than 1μM, exhibiting that these cells were less sensitive to this compound. The activation of IGF-1R and AKT were dose dependently blocked by NVP-AEW541. However, the activities of the key molecules in Ras-MAPK pathway was not significantly inhibited by NVP-AEW541 without any major mutation of Ras. These results indicate that esophageal squamous cell carcinoma may be resistant to targeting IGF-IR due to its maintenance of Ras-MAPK signaling. Thus, to explore the potential mechanism of resistance will contribute to the therapeutic application for NVP-AEW541. Citation Format: Munenori Takaoka, XiaoHong Bao, Huifang Hao, Naomasa Ishida, Takuya Fukazawa, Tomoki Yamatsuji, Minoru Haisa, Yoshio Naomoto. Esophageal cancer exhibits a resistance to the chemical inhibition of IGF-1R with a maintained Ras-MAPK activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2694. doi:10.1158/1538-7445.AM2015-2694

Expression and significance of keratinocyte growth factor and survivin in esophageal squamous cell carcinoma

Objective To investigate the expression of keratinocyte growth factor(KGF) and survivin in human esophageal squamous cell carcinoma(ESCC), and its relationship with clinicopathologic factors of patients with ESCC. Methods Fifty-six cases of surgically resected specimens from ESCC(ESCC gruoup)and 30 cases of normal esophageal epithelia from the broken ends of excisional specimens(the distance to tumor border> 5 cm, control group) were selected for immunohistochemical staining of KGF and survivin expression levels.The relationship of KGF and survivin expression with the clinicopathological features was analyzed. Results The expression levels of KGF and survivin in ESCC group(1. 09±0. 03, and 0. 25±0. 11) were higher significantly than those in control group(4. 11±1. 27,and 0. 36±0. 08,P<0. 05).The expression of KGF in ECSS was correlated with lymph node metastasis and the tumor differentiation degrees(P< 0. 05).The expression of survivin in ECSS was correlated with lymph node metastasis(P<0. 05).There was a positive correlation between KGF and survivin expression in ECSS tissues(r= 0. 873,P< 0. 05). Conclusion KGF and survivin may play important roles in the carcinogenesis and development of ESCC. Key words: Esophageal squamous cell carcinoma; Keratinocyte growth factor; Survivin

Expression and Splice Variant Analysis of Human TCF4 Transcription Factor in Esophageal Cancer.

Objective: The human T cell transcription factor-4 (TCF4) interacts functionally with β-catenin in the Wnt signaling pathway, whose deregulation is involved in the tumorigenesis of various types of cancers. Recent studies showed that TCF4 mRNAs were subject to alternative splicing, which was proposed to be important in regulating transactivational properties of the corresponding protein isoforms. Here we investigated the splicing isoforms and the roles of TCF4 in human esophageal squamous cell carcinoma.Methods: RT-PCR and subsequent cloning and sequencing were applied to identify the splicing isoforms. Western blotting and realtime PCR were used to analyze the expression of TCF4. Knockdown of TCF4 was achieved with siRNA and stable transfection of expression vectors was performed.Results: Our results showed there were a lot of different isoforms of TCF4 mRNA both in human esophageal cancers and cell line. Further, knockdown of TCF4E isoform expression in EC109 cells inhibited the cell growth, while overexpression of TCF4M isoform did not alter its transcription activity. Moreover, sixteen potential binding proteins of TCF4 were preliminarily identified by mass spectrometry.Conclusions: Our data suggested that deregulation of TCF4 isoforms may contribute to the tumorigenesis of ESCC.

Abstract 427: Upregulation of KLF4 and inhibition of HDAC activity by methylseleninic acid in human esophageal squamous cells

Abstract In the present study, we investigated the potential mechanisms of the inhibition of cell growths induced by methylseleninic acid, MSA. Our result showed that MSA could induce cell growth inhibition and apoptosis in esophageal squamous cell carcinoma (ESCC) cells and inhibit tumor growth measured by xenograft volume in nude mice. Upon treated with MSA, HDACs activity was decreased and GCN5, one of histone acetyltransferases, upregulated as well in human esophageal squamous cell lines. Meanwhile a significant increase of H3K9 acetylation (H3K9ac) was detected. Notably,upregulation of KLF4, which has been wildly reported usually downregulated expression in cell lines and tumor tissues of ESCC, was also observed after MSA treatment. Additionally the acetylated histone H3 could locate more at KLF4 promoter region than non-acetylated one did, showed by chromatin immunoprecipitation assay. Moreover knockdown of GCN5 reduced the protein levels of both H3K9ac and KLF4, along with less cell growth inhibition. Taken all, our result indicates that MSA can exert its anti-tumor effects, at least in part, by up-regulation KLF4, probably via inhibiting the HDAC activity and up-regulation GCN5. By our best of knowledge, this is the first report of selenium-induced selective, post-translational acetylation of histone H3 at Lys9, which may depend on the activities of and the balance between HDACs and HATs. Note: This abstract was not presented at the meeting. Citation Format: Chenfei Hu, Wei Zhang, Qing Xu, Lechuang Chen, Kai Ma, Mei Liu, Hongxia Zhu, Ningzhi Xu. Upregulation of KLF4 and inhibition of HDAC activity by methylseleninic acid in human esophageal squamous cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 427. doi:10.1158/1538-7445.AM2014-427