TE-1 Cells Research Articles

Autophagy regulated by JWA influenced sensitivity of esophageal cancer to cisplatin

Objective: To explore the effect of JWA on cisplatin sensitivity and its potential molecular mechanism in esophageal cancer. Methods: The siRNA was used to inhibit the JWA expression, then cisplatin sensitivity and LC3 (autophagy related protein) expression levels were observed in TE1 cells.Further, the effect of autophagy inhibitor tamoxifen (3-MA) on above process was determined.Cisplatin sensitivity of 20 fresh esophageal cancer samples was evaluated by histoculture drug response assay (HDRA). Result: Silencing JWA gene increased the sensitivity of TE1 cells to cisplatin (P<0.05), and decreased the LC3-Ⅰ and LC3-Ⅱ proteins induced by cisplatin.Furthermore, combined with 3-MA increased the inhibition rate of cisplatin in JWA silencing group (P<0.05). Additionally, the inhibition rate of cisplatin on tissues with low JWA expression were higher than those with high expression (45.6% vs 25.6%, P=0.005). Conclusions: JWA could influence the cisplatin sensitivity by regulating autophagy in esophageal cancer.

Krüppel-Like Factor 4 Enhances Sensitivity of Cisplatin to Esophageal Squamous Cell Carcinoma (ESCC) Cells.

BackgroundThe aim of this study was to elucidate the role of Krüppel-Like factor 4 (KLF4) in cisplatin resistance in esophageal squamous cell carcinoma (ESCC) cells, which may eventually help to improve the treatment efficacy.Material/MethodsHuman esophageal squamous cell carcinoma (ESCC) cell line CaEs-17, TE-1, EC109, KYSE510, KYSE140, KYSE70, and KYSE30 were selected to detect their sensitivity to cisplatin. 5-Azacytidine-2′-deoxycytidine (5′-Aza-CdR) treatment and methylation-specific PCR (MS-PCR) were used to detect the methylation status for KLF4. Cell viability, apoptosis, and cell cycle were measured using methyl thiazolyl tetrazolium (MTT) assay, Annexin V affinity assay, and flow cytometry, respectively.ResultsThe sensitivity to cisplatin was different in the seven ESCC cell lines, with TE-1 having the lowest sensitivity and KYSE140 having the highest sensitivity. Interestingly, the level of KLF4 was relatively low in TE-1 cells; while it was high in KYSE140 cells. These results suggested that KLF4 may be involved in cisplatin resistance. The promoter region was mostly unmethylated in KYSE140 cells; while it was hypermethylated in TE-1 cells. After treatment with demethylation reagent 5-Aza-CdR, cisplatin sensitivities were significantly increased after upregulation of KLF4, as the IC50 values were significantly decreased in the TE-1 cell treated with 5-Aza-CdR. Furthermore, upregulation of KLF4 induced cell apoptosis and cell cycle arrest at S phase.ConclusionsKLF4 enhances the sensitivity of cisplatin to ESCC cells through apoptosis induction and cell cycle arrest. Our data provided a novel insight to the mechanism of cisplatin resistance; overexpression of KLF4 may be a potential therapeutic strategy for cisplatin resistance in human ESCC.

Derlin-1 is a target to improve radiotherapy effect of esophageal squamous cell carcinoma.

Radiotherapy is widely used for treatment of esophageal squamous cell carcinoma (ESCC). This study aimed to explore the role of Derlin-1 on the sensitivity of ESCC to radiotherapy and its underlying mechanism. We examined the clinical significance of Derlin-1 in 125 ESCC tissues. We found that Derlin-1 protein was higher in ESCC tissues than that in normal esophageal epithelial tissues. Derlin-1 overexpression was correlated with chemoradiotherapy resistance in ESCC patients and served an independent predictor for short overall survival. siRNA knockdown and plasmid transfection were carried out in ESCC cell lines. Derlin-1 depletion inhibited cell growth while its overexpression facilitated cell growth. Derlin-1 overexpression in Eca-109 cells dramatically enhanced its resistance to radiotherapy with decreased apoptosis rate. On the contrary, Derlin-1 depletion in TE-1 cell line showed the opposite effects. In addition, radioresistance conferred by Derlin-1 was attributed to its role of activating AKT/Bcl-2 signaling pathway and reducing caspase3 cleavage. Blockage of AKT signaling attenuated the role of Derlin-1 on radioresistance. Furthermore, Derlin-1 could interact with PI3K p110α in ESCC cell lines. Taken together, Our data demonstrate that Derlin-1 overexpression predicts poor prognosis and protects ESCC from irradiation induced apoptosis through PI3K/AKT/Bcl-2 signaling pathway. Derlin-1 may serve as a novel predictor for radiosentivity and a molecular target for ESCC.

Abstract 4407: Glucose transporter 1 is associated with proliferation and prognosis on esophageal squamous cell carcinoma

Abstract Purpose: Cancer cells exhibit altered glucose metabolism, termed glycolysis which is described by the increased uptake of glucose and the conversion of glucose to lactate in cancer cells under adequate oxygen tension. Alternating glucose transporters (GLUTs) expression was detected in the cancer specific metabolism to adapt to the rapid growth and tumor microenvironment in diverse malignant tumors. In this study, GLUTs profiling of ESCC patients, the relation between proliferation and GLUT1 expression using siRNA for GLUT1 were analyzed. Furthermore, the prognostic features according to GLUT1 expression was evaluated on esophageal squamous cell carcinoma (ESCC) patients. Methods: The ratio of the 11 GLUTs (GLUT1- GLUT14) expression between tumor and normal tissue (T/N) were analyzed in silico (GDS3838). GLUT1 expression was down-regulated using 3 siRNAs in vitro. Immunohistochemical staining of GLUT1 was performed using paraffin sections of tissues obtained from 145 resectable ESCC patients without preoperative treatment. Results: Expression of GLUT1 (T/N 2.44; 95% confidence interval (CI): 1.78-3.34, P&amp;lt;0.001), GLUT3 (T/N 1.96; 95%CI: 1.23-3.13, P=0.01) and GLUT6 (T/N 1.31; 95%CI: 1.12-1.54, P&amp;lt;0.001) increased in tumor tissue compared with normal tissue. TE-1 and TE-8 cells were treated with 3 siRNAs for GLUT1 and control siRNA for 72 hours in vitro. The proliferation of TE-1 and TE-8 cells after siRNAs for GLUT1 compared with control siRNA decreased 72.9-84.5% and 44.8-53.3%, respectively. GLUT1 expression was evaluated with 145 ESCC patients. Glut1 positivity was observed in 41 patients (28.2%) and associated with depth of invasion (odds ratio (OR)=2.984; 95%CI: 1.208-7.371, P=0.018) and vascular invasion (OR=2.771; 95% CI: 1.118-6.871, P=0.028) in multivariate analysis. GLUT1 positivity was a significant disadvantage to both relapse-free survival (hazard ratio (HR)=2.021; 95%CI: 1.100-3.712; P=0.023) and esophageal cancer-specific survival (HR=2.223; 95%CI: 1.121-4.411; P=0.022) in univariate Cox hazard analysis. Conclusions: GLUTs expression was altered in ESCC patients and GLUT1 expression was most up-regulated among the 11 GLUTs. Growth assay with siRNAs for GLUT1 revealed that GLUT1 played a curial role of ESCC cells proliferation. High expression of GLUT1 was associated with depth of invasion and poor prognosis of ESCC patients. These findings provide evidence of the significance of GLUT1 expression as a biomarker and GLUT1 may be therapeutic target in ESCC patient. Citation Format: Hiroshi Sawayama, Masaaki Iwatsuki, Naoya Yoshida, Yoshifumi Baba, Yasuo Sakamoto, Koichi Kinoshita, Kenichi Nakamura, Daisuke Kuroda, Eto Tsugio, Tasuku Toihata, Tomoyuki Uchihara, Eri Oda, Taisuke Yagi, Mayuko Ohuchi, Takatugu Ishimoto, Hideo Baba. Glucose transporter 1 is associated with proliferation and prognosis on esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4407. doi:10.1158/1538-7445.AM2017-4407

Role of HDM2 Gene in Radio-Sensitivity of Esophageal Cancer Cell Lines to Irradiation

Background: Radio therapy plays an important role in controlling tumor growth in esophageal cancer patients. Objectives: Our study provided comprehensive information about radio-sensitivity of Hdm2 gene in esophageal cancer cells in cancer cells. Methods: The expression of Hdm2 gene in radiosensitive and radio-resistant esophageal squamous cell carcinoma TE1, TE8 and TE11 cell lines is studied. RNA extracted from each esophageal cancer cell, then Quantitative reverse transcription PCR (RT-qPCR) assay were used for measuring HDM2 gene expression. Finally, radiosensitivity was determined for every three esophageal cancer cell lines. Results: It was found that Hdm2 gene significantly expressed in TE1, TE8 and TE11 cell lines by qRT-PCR analysis. Clonogenic assay showed that TE-11 was very sensitive to radiation relative to other cell lines. Conclusions: Hdm2 protein binding to the P53 protein is a key regulator of cell cycle control and ‎genome protection Cell cycle control, programmed cell death and response cells to ‎radition play important roles.‎

2-Acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino) phenyl carbamoylsulfanyl] propionic acid, a glutathione reductase inhibitor, induces G2/M cell cycle arrest through generation of thiol oxidative stress in human esophageal cancer cells.

Esophageal squamous cell carcinoma (ESCC) is a highly malignant cancer with poor response to both of chemotherapy and radiotherapy. 2-Acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino) phenyl carbamoylsulfanyl] propionic acid (2-AAPA), an irreversible inhibitor of glutathione reductase (GR), is able to induce intracellular oxidative stress, and has shown anticancer activity in many cancer cell lines. In this study, we investigated the effects of 2-AAPA on the cell proliferation, cell cycle and apoptosis and aimed to explore its mechanism of action in human esophageal cancer TE-13 cells. It was found that 2-AAPA inhibited growth of ESCC cells in a dose-dependent manner and it did not deplete reduced glutathione (GSH), but significantly increased the oxidized form glutathione (GSSG), resulting in decreased GSH/GSSG ratio. In consequence, significant reactive oxygen species (ROS) production was observed. The flow cytometric analysis revealed that 2-AAPA inhibited growth of esophageal cancer cells through arresting cell cycle in G2/M phase, but apoptosis-independent mechanism. The G2/M arrest was partially contributed by down-regulation of protein expression of Cdc-25c and up-regulation of phosphorylated Cdc-2 (Tyr15), Cyclin B1 (Ser147) and p53. Meanwhile, 2-AAPA-induced thiol oxidative stress led to increased protein S-glutathionylation, which resulted in α-tubulin S-glutathionylation-dependent depolymerization of microtubule in the TE-13 cells. In conclusion, we identified that 2-AAPA as an effective thiol oxidative stress inducer and proliferation of TE-13 cells were suppressed by G2/M phase cell cycle arrest, mainly, through α-tubulin S-glutathionylation-mediated microtubule depolymerization. Our results may introduce new target and approach for esophageal cancer therapy through generation of GR-mediated thiol oxidative stress.

Inhibitory effects of microRNA-143 on invasion and metastasis of esophageal cancer by down-regulating Wnt/β-catenin and mechanism

Objective To discuss the inhibitory effects of microRNA (miRNA, miR)-143 on the invasion and metastasis of esophageal cancer (EC) cells by downregulating Wnt/β-catenin level. Methods The EC109-H, TE-1 and Het-1A cells were cultured in vitro. Then the cells were randomly divided into experimental group (EC109-H, TE-1)and negative control group (Het-1A); Transwell assay, real-time quantitative polymerase chain reaction(Real-time PCR) and Western blotting were used to detect invasive ability of each group, and the expression of miR-143, Wnt, β-catenin and CD44 related to signaling pathway Wnt/β-catenin in different cell lines. The correlation between miR-143 and regulatory proteins of Wnt/β-catenin was analyzed. The targets related to miR-143 were predicted through online software. The miR-143 inhibitors and mimics were used to change the levels of miR-143 in each group, and CD44-3’untranslated region (3’UTR) carrier and CD44-3’untranslated regions mutation (3’UTRM) mutant carrier were verified. The forecast target was vertified by the double luciferase experiment. Results Of the cell lines, EC109-H had the strongest invasive ability (156.86±0.83, P=0.003). The expression levels of miR-143 in EC109-H cells were the lowest (1.01±0.12, P=0.000). The expression levels of Wnt, β-catenin and CD44 in EC109-H cells which had strongest invasive ability were the highest (0.51±0.11, P=0.005; 0.65±0.12, P=0.007; 0.80±0.09, P=0.001). Pertinence analysis showed there was a negative correlation between miR-143 and those proteins (rCD44=-0.827, P=0.000; rwnt=-0.907, P=0.017; rβ-catenin=-0.810, P=0.028). We predicted CD44 might be a control target of miR-143. Dual luciferase assays showed that miR-143 could target CD44-3’UTR region (0.32±0.28, P=0.000). Conclusion MiR-143 may regulate the migration and invasion ability of esophageal cancer cells by targeting Wnt/β-catenin signaling pathway. Key words: MicroRNA-143; Wnt/β-catenin signaling pathway; Esophageal cancer; Invasion; Metastasis

Programmed death-ligand 1 expression at tumor invasive front is associated with epithelial-mesenchymal transition and poor prognosis in esophageal squamous cell carcinoma.

Programmed death‐ligand 1 (PD‐L1) plays a crucial role in the host immune system in cancer progression. The gene promoter region of PD‐L1 also contains a binding site for ZEB1, a transcription factor related to epithelial‐mesenchymal transition (EMT). The purpose of this study was to clarify the relationship between PD‐L1 and EMT and its clinical importance in esophageal squamous cell carcinoma (ESCC). PD‐L1 and ZEB1 expression at the tumor invasive front was examined by immunohistochemistry in resected specimens from 90 patients with ESCC who underwent surgery without preoperative therapy, and their expression and clinicopathological factors were compared. ZEB1 and PD‐L1 expression was determined in TE8 cells, which demonstrate the EMT phenotype, following ZEB1 knockdown by siZEB1. TE5, TE6 and TE11 cells with non‐EMT phenotype were also used for studies of TGF‐β1‐dependent EMT induction and ZEB1 and PD‐L1 expression. In cases of high PD‐L1 expression at the invasive front, significantly greater depth of tumor invasion, EMT, and less CD8+ lymphocyte infiltration were observed. High PD‐L1 expression was also associated with worse overall and relapse‐free survival. A correlation was observed between PD‐L1 and ZEB1 expression. In TE8 cells, siZEB1 suppressed PD‐L1 and promoted E‐cadherin mRNA and protein expression. TGF‐β1 induced EMT and surface expression of PD‐L1 in TE5, TE6 and TE11 cell lines. PD‐L1 expression at the ESCC invasive front was related to ZEB1 expression, EMT and poor prognosis. We suggest that a cooperative mechanism bridging between tumor immune avoidance and EMT contributes to tumor malignancy in ESCC.

Association of epithelial-mesenchymal transition with an immunosuppressive tumor microenvironment with elevated levels of PD-L1 in esophageal carcinoma.

e15585 Background: Programmed death-ligand 1 (PD-L1) plays a crucial role in the host immune system in cancer progression and is has become a new target for cancer therapy. PD-L1 gene promoter region contains a binding site of ZEB1, a transcription factor related to epithelial-mesenchymal transition (EMT). The purpose of this study is to clarify relationships between PD-L1, EMT and its clinical importance in esophageal squamous cell carcinoma (ESCC). Methods: We conducted preclinical studies using cell lines and clinical specimens. ZEB1, PD-L1 expression in TE8 cell line, demonstrating EMT phenotype, was determined following ZEB1 knockdown by siZEB1. TE5, 6 and 11 cell lines with non-EMT phenotype were also used for studies of TGF-β1-dependent EMT induction and ZEB1, PD-L1 expression. PD-L1 and ZEB1 expression at the tumor invasive front was examined in resected specimens from 90 patients with ESCC who underwent surgery without preoperative therapy by immunohistochemistry and their expression and clinicopathological factors were compared. Results: A correlation was observed between PD-L1 and ZEB1 expression. In TE8 cells which express EMT character , siZEB1 suppressed PD-L1 and promoted E-cadherin mRNA and protein expression. In TE5, 6 and 11 cell lines which are not EMT phenotype, TGF-β1 induced EMT and surface expression of PD-L1. In cases of high PD-L1 expression at invasive front, greater depth of tumor invasion, EMT, less CD8 expression were significantly observed. High PD-L1 expression was also associated with worse overall and relapse-free survival. Conclusions: PD-L1 expression at the invasive front was related to ZEB1 expression, EMT and poor prognosis in ESCC. We suggest that cooperative mechanism between tumor immune avoidance and EMT contributes to tumor malignancy. Whether or not ZEB1-PD-L1 signal pathway could be a target in treatment for ESCC requires further investigations.

BRAF-activated non-protein coding RNA (BANCR) advances the development of esophageal squamous cell carcinoma via cell cycle

AbstractEsophageal carcinoma is one of the important lethal tumors in the world, but the mechanism of esophageal cancer progression is still unclear. The long non-coding RNAs are known to regulate tumor progression. Here we report that BRAF-activated nonprotein coding RNA (BANCR) is associated with the development of esophageal squamous cell carcinoma (ESCC). The expression levels ofBANCRin 132 ESCC tumor tissues detected by quantitative real-time PCR were significantly higher than that in the adjacent normal tissues. The expression level ofBANCRwas down-regulated by transfecting small interrupting RNA againstBANCR(BANCRsiRNA) in Eca109 cells. Eca109 and TE-1 cells transfected withBANCRsiRNA led to the delayed cell cycles compared to the cells transfected with negative control siRNA. Moreover, MTT assays demonstrated that Eca109 cells transfected withBANCRsiRNA resulted in the inhibition of cell proliferation. These results suggested thatBANCRadvances the development and progression of ESCC cells through regulating their cell cycles.

Decreased expression of CD63 tetraspanin protein predicts elevated malignant potential in human esophageal cancer.

The tetraspanin CD63 has been described to have critical roles in multiple biological processes, including tumorigenesis and metastasis in several types of cancer. However, its role in esophageal carcinoma (EC) has not been reported. In the current study, immunohistochemistry was used to investigate CD63 expression in 106 esophageal cancer samples, 49 adjacent esophagus tissues and 17 normal esophagus mucosa tissues. The results revealed that the overexpression of CD63 was observed in esophageal cancer samples and negatively correlated with tumor stage and lymph node metastasis. To further evaluate the role of CD63 in esophageal carcinoma, the invasiveness of EC cells was analyzed using matrigel invasion assays and wound healing assays in vitro. Furthermore, it was found that CD63 knockdown increased the invasiveness of TE-1 cells through the upregulation of matrix metalloproteinase (MMP) expression via promoting epithelial-mesenchymal transition. The current data therefore suggested that low levels of CD63 expression may be involved in the tumor progression of esophageal carcinoma.

Esophageal cancer stem cells are suppressed by tranilast, a TRPV2 channel inhibitor.

Recent evidence suggests that the targeting of membrane proteins specifically activated in cancer stem cells (CSCs) is an important strategy for cancer therapy. The objectives of the present study were to investigate the expression and activity of ion-transport-related molecules in the CSCs of esophageal squamous cell carcinoma. Cells exhibiting strong aldehyde dehydrogenase1 family memberA1 (ALDH1A1) activity were isolated from TE8 cells by fluorescence-activated cell sorting, and CSCs were then generated with the sphere formation assay. The gene expression profiles of CSCs were examined by microarray analysis. Among TE8 cells, ALDH1A1 messenger RNA and protein levels were higher in CSCs than in non-CSCs. The CSCs obtained were resistant to cisplatin and had the ability to redifferentiate. The results of the microarray analysis revealed that the expression of 50 genes encoding plasma membrane proteins was altered in CSCs, whereas that of several genes related to ion channels, including transient receptor potential vanilloid 2 (TRPV2), was upregulated. The TRPV2 inhibitor tranilast was more cytotoxic at a lower concentration in CSCs than in non-CSCs, and effectively decreased the number of tumorspheres. Furthermore, tranilast significantly decreased the cell population that strongly expressed ALDH1A1 among TE8 cells. The results of the present study suggest that TRPV2 is involved in the maintenance of CSCs, and that its specific inhibitor, tranilast, has potential as a targeted therapeutic agent against esophageal squamous cell carcinoma.

Downregulation of TNFAIP2 suppresses proliferation and metastasis in esophageal squamous cell carcinoma through activation of the Wnt/β-catenin signaling pathway.

Tumor necrosis factor-α (TNF-α) plays a pivotal role in malignant tumor formation in the tumor microenvironment. To investigate the role of TNF-α in esophageal squamous cell carcinoma (ESCC), we assessed expression profiles of the downstream gene TNF-α-induced protein2 (TNFAIP2), which e previously unknown in ESCC. TNFAIP2 mRNA and protein expression levels were examined by qRT-PCR and immunohistochemical analysis in 24fresh and 55paraffin‑embedded specimens, respectively. The results demonstrated that TNFAIP2 mRNA and protein levels were overexpressed in tumor cells, and TNFAIP2 overexpression was significantly associated with Tstage (p=0.049), Nstage (p=0.019) and the International Union Against Cancer (UICC) stage (p=0.028). Invitro, TNFAIP2 was highly expressed in TNFα-stimulated Eca109, Kyse150, Kyse510 and TE-10 cells. Lentivirus-mediated RNA interference of TNFAIP2 inhibited cell proliferation, colony formation, migration, invasion and the cell cycle. Moreover, LV-RNAi-mediated TNFAIP2 was found to regulate the Wnt/β-catenin by decreasing expression of some genes downstream from β-catenin (i.e., C-myc, cyclinD1, MMP-7 and Snail), and upregulating expression of E-cadherin and p-GSK-3β. Taken together, these results show that TNFAIP2 may be a potential tumorigenesis gene in ESCC. Our data indicate that TNFAIP2 overexpression may facilitate proliferation and metastasis via activation of the Wnt/β-catenin signaling pathway in ESCC.

Nutlin-3, an Antagonist of MDM2, Enhances the Radiosensitivity of Esophageal Squamous Cancer with Wild-Type p53

Murine double minute 2 (MDM2) negatively regulates the activity of the p53 protein and plays a vital role in cell cycle arrest, apoptosis, and senescence mediated by p53. Nutlin-3, an antagonist of MDM2, is frequently used in anti-cancer studies. In many human tumors, nutlin-3 stabilizes p53 status and enhances p53 expression in cells with wild-type p53. However, the effect of nutlin-3 combined with radiotherapy on esophageal squamous cancer (ESCC) has not been reported. In this study, we examined whether nutlin-3 increases the radiosensitivity of ESCC in vitro and in vivo.We chose two cell lines, ECA-109 (wild-type p53) and TE-13 (p53 mutated), for the following experiments. Cell proliferation and clonogenic survival experiments showed that nutlin-3 inhibits the cell growth and colony formation of ECA-109 cells in a dose-dependent manner. Flow cytometry analysis showed that the apoptosis rate of ECA-109 cells co-treated with nutlin-3 and irradiation(IR) was significantly increased compared with cells treated with irradiation or nutlin-3 alone. Western blotting detected the expression of apoptosis-associated proteins in ECA-109 cells in response to nutlin-3 and irradiation. These effects were not evident in TE-13 cells. Xenograft mouse models indicated that nutlin-3 suppresses tumor growth and promotes radiosensitivity in the ESCC cell line ECA-109 in vivo. We have demonstrated that co-treatment of nutlin-3 with irradiation can significantly inhibit the growth and improve the radiosensitivity of ESCC cells with wild-type p53. The study suggests that nutlin-3 may be a potent therapeutic agent in conjunction with radiotherapy in ESCC.

Effect of SATB1 silencing on the proliferation, invasion and apoptosis of TE-1 esophageal cancer cells.

The aim of the present study was to investigate the effect of special AT-rich sequence-binding protein-1 (SATB1)-targeted small interfering RNA (siRNA) on the proliferation, invasion and apoptosis of TE-1 human esophageal cancer cells. SATB1 has been correlated with the metastasis and poor prognosis of colon and breast cancer, but the role of SATB1 in esophageal cancer remains unknown. Therefore, the present study constructed and transfected SATB1-siRNA into TE-1 cells in order to knockdown the expression of the SATB1 gene. Western blot analysis, a cell counting kit, transwell chamber assays and flow cytometry were used to assess the effect of SATB1-siRNA on the proliferation, invasion and apoptosis of cells. The results demonstrated that the expression of the SATB1 gene was efficiently knocked down by SATB1-siRNA, and that SATB1-siRNA inhibited the proliferation, invasion and apoptosis of TE-1 cells. Therefore, it was concluded that the SATB1 gene is important in the pathogenesis of human esophageal cancer, and may present a novel therapeutic target for esophageal cancer.

Musashi1, a potential prognostic marker in esophageal squamous cell carcinoma.

Esophageal cancer ranks as the sixth leading cause of cancer-related deaths worldwide. Cancer stemness is mainly considered to be the key factor for cancer recurrence particularly in esophageal cancer. It is important to identify cancer stem cell markers as targets in future therapies. The present study aimed to investigate the expression of putative cancer stem cell-related marker musashi1 (Msi1) and assess the correlation with clinicopathologcal status of esophageal squamous cell carcinoma (ESCC) cases. We then clarified the role of Msi1 in esophageal cancer cells during proliferation, apoptosis, sphere formation and migration. Finally, we investigated the relationship of Msi1 with the prognosis of ESCC patients. ESCC tissue samples from 93 patients and 20 paired histologically normal tissues were procured for immunohistochemical analysis. We analyzed the characteristics of Msi1, using sphere formation and anchorage independent growth. Moreover, using flow cytometry and Cell Counting Kit-8 (CCK-8) assay, we investigated the role of Msi1 in cancer cell proliferation and apoptosis. Furthermore, we clarified the role of Msi1 in the process of sphere formation and migration of ESCC cells through knockdown of Msi1 expression by siRNA in ESCC cell lines. The results revealed that there was a higher expression of Msi1 in ESCC specimens compared with normal tissues. In addition, Msi1 expression was significantly associated with clinical stage and lymph node metastasis. Most importantly, the increased immunocytochemical staining of Msi1 in spheroid cells revealed the stemness characteristics of Msi1 in ESCC. In addition, we found that silencing of Msi1 decreased cell proliferation, migration and induced apoptosis in TE-7 and KYSE70 cells. Furthermore, downregulation of Msi1 attenuated the sphere formation ability of ESCC cells. Patients with higher expression of Msi1 had a shorter survival. In conclusion, Msi1 acts as a stemness-associated gene in esophageal cancer cell lines and could serve as a prognostic marker in patients with ESCC.

Downregulation of HuR Inhibits the Progression of Esophageal Cancer through Interleukin-18.

PurposeThe purpose of this study was to investigate the effect of human antigen R (HuR) downregulation and the potential target genes of HuR on the progression of esophageal squamous cell carcinoma (ESCC).Materials and MethodsIn this study, a proteomics assay was used to detect the expression of proteins after HuR downregulation, and a luciferase assay was used to detect the potential presence of a HuR binding site on the 3’-untranslated region (3'-UTR) of interleukin 18 (IL-18). In addition, colony formation assay, MTT, EdU incorporation assay, Western blot, flow cytometry, immunohistochemistry, transwell invasion assay, and wound healing assay were used.ResultsIn the present study, we found that the expression of both HuR protein and mRNA levels were higher in tumor tissues than in the adjacent tissues. HuR downregulation significantly suppressed cell proliferation. In addition, the metastasis of esophageal cancer cells was inhibited, while the expression of E-cadherin was increased and the expression of matrix metalloproteinase (MMP) 2, MMP9, and vimentin was decreased after HuR knockdown. Moreover, silencing of HuR disturbed the cell cycle of ESCC cells mainly by inducing G1 arrest. Furthermore, proteomics analysis showed that downregulation of HuR in TE-1 cells resulted in 100 upregulated and 122 downregulated proteins, including IL-18 as a significantly upregulated protein. The expression of IL-18 was inversely regulated by HuR. IL-18 expression was decreased in ESCC tissues, and exogenous IL-18 significantly inhibited the proliferation and metastasis of ESCC cells. The 3'-UTR of IL-18 harbored a HuR binding site, as shown by an in vitro luciferase assay.ConclusionHuR plays an important role in the progression of esophageal carcinoma by targeting IL-18, which may be a potential therapeutic target for the treatment of ESCC.

STAT3 down-regulation induces mitochondria-dependent G2/M cell cycle arrest and apoptosis in oesophageal carcinoma cells.

STAT3 is persistently activated in a wide variety of human tumours, and aberrant STAT3 activity promotes tumour growth, invasion and metastasis. To explore STAT3 down-regulation in human oesophageal cancer cells, cell proliferation, apoptosis and mitochondrial mechanisms were explored in oesophageal carcinoma TE1 cell cultures. We demonstrate for the first time that STAT3 down-regulation by RNAi is sufficient to inhibit oesophageal cancer cell proliferation inducing cell apoptosis. Further, we demonstrate that mitochondrial transmembrane potential is impaired thereby leading to collapsed mitochondrial membrane potential, abnormal mitochondrial membrane depolarization, nuclear DNA fragmentation and cell cycle G2/M arrest under the conditions of STAT3 down-regulation. Thus, our results suggest that STAT3 inhibition is a valid approach to induce oesophageal carcinoma cell mitochondrial-dependent apoptosis in therapeutic strategies against oesophageal cancers.

MiR-30b inhibits cancer cell growth, migration, and invasion by targeting homeobox A1 in esophageal cancer

Emerging evidence has shown that microRNAs (miRNAs) play important roles in tumor development and progression. In particular, miR-30b is thought to be closely related to the migration, invasion, proliferation, communication, and drug resistance of tumor cells. However, the potential value of miR-30b in human esophageal cancer (EC) remains unclear. In this study, we investigated the biological functions of miR-30b and its potential role in EC. The results indicated that the expression levels of miR-30b were decreased in EC tissues and were correlated with invasion classification (P < 0.01), lymph node metastasis (P < 0.01), and pathological stage (P < 0.05). Log-rank tests demonstrated that low expression of miR-30bwas strongly correlated with poor overall survival in patients with EC (P < 0.05). Moreover, overexpression of miR-30b markedly inhibited the growth, migration, and invasion of ECA109 and TE-1 cells by directly downregulating homeobox A1 (HOXA1). When HOXA1 was reintroduced into miR-30b-transfected ECA109 or TE-1 cells, the inhibitory effects of miR-30b on EC cell growth, migration, and invasion were markedly reversed. In conclusion, our findings demonstrated that miR-30b could inhibit tumor cell growth, migration, and invasion by directly targeting HOXA1 in EC cells.

TNFAIP3 overexpression is an independent factor for poor survival in esophageal squamous cell carcinoma.

Tumor necrosis factor α induced protein3 (TNFAIP3) is a protein that is induced by TNF-mediated NF-κB activation and has a dual function in regulating NF-κB. TNFAIP3 is associated with inflammatory carcinogenesis in many cancer types. However, the clinical significance of TNFAIP3 expression and function in esophageal squamous cell carcinoma (ESCC) has not yet been reported. We examined 149ESCC tissue specimens to determine the clinical significance of TNFAIP3 by immunohistochemistry. Western blot analyses were used to detect TNFAIP3 expression in TE-1, TE-8, TE-15 and KYSE-70 ESCC cells and in Het-1A, a non-cancerous esophageal cell line. TNFAIP3 protein knockdown was conducted using small-interfering RNA to investigate its impact on cell proliferation, migration and invasion. Significant correlations between TNFAIP3 expression and differentiation (P=0.04) among clinicopathological characteristics of ESCC patients were demonstrated, and high TNFAIP3 expression was associated with poor survival (P=0.02). Moreover, multivariate analysis result showed that high TNFAIP3 expression was an independent factor for poor survival (P=0.04). In vitro analysis showed high expression of TNFAIP3 protein in TE-15 cells and low expression in Het-1A cells. Furthermore, the proliferation, migration and invasion of TE-15 cells after TNFAIP3 suppression by siRNA were significantly reduced. These findings suggest that TNFAIP3 protein may be an independent prognostic marker for poor survival, and a promising target for ESCC therapy.