Esophageal Squamous Cell Carcinoma EC9706 Research Articles

Pigment Epithelium-Derived Factor Promotes the Growth and Migration of Human Esophageal Squamous Cell Carcinoma

Pigment epithelium-derived factor (PEDF) is an oncogene found in various types of cancers. However, how PEDF affects the development of human esophageal squamous cell carcinoma (ESCC) is unknown. This study investigates the role of PEDF in ESCC cell proliferation, migration, and cell cycle both in vitro and in vivo. The PEDF expression was examined in patient tumor samples and ESCC cell lines. Short hairpin RNA technology was used to inhibit the PEDF expression in ESCC EC9706 and KYSE150 cells. In vitro cell proliferation and migration assays were performed. The effects of PEDF on tumor growth and progression were examined in vivo in murine subcutaneous xenograft tumor models. It was found that PEDF was overexpressed in esophageal cancer cells and patient tumor tissues compared to normal control samples. PEDF enhanced cell cycle progression and inhibited cell apoptosis. Knock down of PEDF inhibited esophageal cell proliferation and migration in vitro. Moreover, Inhibition of PEDF significantly reduced tumor growth and tumor size in vivo. These results indicate that PEDF induce tumorigenesis in ESCC and can be a potential therapeutic target for cancer treatment.

C-Phycocyanin elicited antitumor efficacy via cell-cycle arrest, apoptosis induction, and invasion inhibition in esophageal squamous cell carcinoma

Objectives: Mounting evidence has demonstrated that C-Phycocyanin (C-PC) exhibits marked antitumor activity in a wide type of tumors, such as pancreas cancer, breast carcinoma, lung cancer, and colon cancer. The current study aimed to confirm the antitumor efficacy of C-PC in esophageal squamous cell carcinoma (ESCC).Methods: The efficacy of C-PC was evaluated against the proliferation of ESCC cell lines EC9706 and EC1 by CCK-8 kit and in a mice model of ESCC EC9706. Cell cycle and apoptosis were investigated by flow cytometry, and cell invasion was determined via transwell chamber. Protein expression was examined by Western blots.Results: We found that C-PC exhibited anti-proliferation ability in a time-dependent manner and a dose-dependent manner in ESCC EC9706 and EC1 cells. Besides, C-PC markedly arrested cell cycle in the G0/G1 phase, induced cell apoptosis and suppressed cell invasion ability in both EC9706 and EC1 cells (p < .01). Notably, C-PC evoked the elevations of Bax, PARP, and cleaved-caspase-3 protein, but reduced cyclin D1, CDK4, Bcl-2, MMP-2, and MMP-9 expression levels. Further investigation from in vivo experiment revealed that C-PC displayed significant antitumor efficacy in the xenografted EC9706 model.Conclusions: Our data presented herein suggest C-PC exerts antitumor efficacy in ESCC.

Mechanisms of the suppression of proliferation and invasion ability mediated by microRNA-147b in esophageal squamous cell carcinoma

Objective: To explore the expression of microRNA(miR)-147b in esophageal squamous cell carcinoma (ESCC) and its regulatory roles in cell proliferation, cell cycle and invasion as well as its molecular mechanisms. Methods: Real-time quantitative PCR (qPCR) was used to investigate the expression of miR-147b in ESCC tissues and cells. Negative control (NC) and miR-147b inhibitor were transfected into ESCC EC1 and EC9706 cells, which were divided into two groups: NC group and miR-147b inhibitor group, and qPCR was employed to detect the miR-147 level and CCK-8. Flow cytometry and Transwell chamber were utilized to investigate the effects of miR-147b downregulation on cell proliferation, cell cycle and invasion in ESCC cells. Besides, target genes of miR-147b was confirmed by double luciferase reporter assay. Subsequently, qPCR and Western blot were used to examine the effects of miR-147b downregulation on NDUFA4 expression, and NDUFA4 expression and its correlation with miR-147b were investigated in ESCC tissues. Results: Relative level of miR-147b in ESCC tissues (3.03±0.27) and cells were markedly higher than that in para-carcinoma tissues (1.00±0.01) and normal esophageal epithelial cell, and the differences had statistical significance (P<0.01), and its high expression was closely associated with clinical staging, invasion depth, histological grading and lymph node metastasis(P<0.05). Importantly, clinical staging, lymph node metastasis and miR-147b may be an independent prognostic factor in ESCC. Moreover, miR-147b downregulation dramatically suppressed cell proliferation, arrested cell cycle in G0/G1 phase and reduced invasion ability in ESCC cells. Most importantly, NDUFA4 was a direct target gene of miR-147b, and miR-147b inhibitor evidently upregulated the expression of NDUFA4. Furthermore, NDUFA4 displayed low expression in ESCC tissues and its expression exhibited negative correlation with miR-147b expression. Conclusions: The downregulation of miR-147b expression significantly suppresses the proliferation and invasion abilities as well as alters cell cycle distribution in ESCC.

MicroRNA-125a-5p enhances the sensitivity of esophageal squamous cell carcinoma cells to cisplatin by suppressing the activation of the STAT3 signaling pathway.

Increasing evidence has demonstrated that microRNAs (miRNAs or miRs) play a variety of roles in tumor development, progression and chemosensitivity in a wide range of tumors. In this study, we found that miR-125a-5p exhibited a low expression in esophageal squamous cell carcinoma (ESCC) tissues and cells, and that its low expression was associated with higher tumor staging and shorter a survival time of patients with ESCC. Moreover, miR-125a-5p overexpression contributed to the suppression of cell proliferation, cell cycle arrest, cell apoptosis and a decrease in cell migratory and invasive abilities, whereas the downregulation of miR-125a-5p promoted cell proliferation, accelerated cell cycle progression, suppressed apoptosis and enhanced the migratory and invasive abilities of ESCC EC1 and TE1 cells, which may be tightly associated with the epithelial-mesenchymal transition (EMT) process in ESCC. Importantly, miR-125a-5p enhanced the cytotoxic effects of cisplatin on EC1 and TE1 cells, and co-treatment with miR-125a-5p and cisplatin significantly induced cell apoptosis and reduced the cell migratory and invasive abilities of EC1 and TE1 cells, coupled with an increase in the E-cadherin level and a decrease in the N-cadherin and Vimentin levels. Most notably, signal transducer and activator of transcription-3 (STAT3) was found to be a direct target of miR-125a-5p in ESCC cells, and miR-125a-5p overexpression significantly reduced the protein levels of t-STAT3, p-STAT3 and vascular endothelial growth factor (VEGF) in EC1 and TE1 cells. Furthermore, the combination of miR-125a-5p and cisplatin markedly inactivated the STAT3 signaling pathway; however, interleukin (IL)-6, a widely reported activator of the STAT3 signaling pathway, reversed the suppressive effects of miR-125a-5p/cisplatin in ESCC cells on the activation of the STAT3 signaling pathway. Of note, we found that IL-6 markedly reversed the altered cell phenotype mediated by the combination of miR-125a-5p and cisplatin in ESCC cells. These findings suggest that miR-125a-5p may play a pivotal role in the development and progression of ESCC, which may be achieved via the manipulation of the STAT3 signaling pathway.

Function of miR-25 in the invasion and metastasis of esophageal squamous carcinoma cells and bioinformatical analysis of the miR-106b-25 cluster.

MicroRNAs (miRNAs/miRs) are a class of small, non-coding RNA molecules that serve a key function in carcinogenesis and tumor progression. Recent evidence indicates that miRNAs may act as powerful regulators of migration and invasion. The present study aimed to investigate the effect of miR-25 on the invasion and metastasis of KYSE-150 and EC109 esophageal squamous cell carcinoma (ESCC) cells, and predict the mechanism of this effect by bioinformatically analyzing the miR-106b-25 cluster. In order to alter the expression of miR-25 in the two cell lines, a miR-25 inhibitor or mimic were transfected into the cells, which were then studied via Transwell migration and invasion assays. Subsequently, the target genes of the miR-106b-25 cluster were predicted using miRanda, PicTar, TargetScan and miRTarbase, and the functions of the target genes were predicted via Gene Ontology term and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. Then, a protein-protein interaction (PPI) network was produced using the Search Tool for the Retrieval of Interacting Genes. The results revealed that overexpressing miR-25 led to significantly increased cell migration and invasion in KYSE150 and EC109 cells. Suppressing miR-25 resulted in significantly decreased cell migration and invasion in KYSE150 cells, while the result was not significant in EC109 cells. Target genes of the miR-106b-25 cluster were significantly enriched in the biological process regulation of cellular metabolic process and several cancer-associated pathways, such as those for glioma and melanoma. The PPI network revealed that PTEN, TP53, MDM2, E2F1, PRMT5, MCM2, RB1, CDKN1A, SHAD7 and EZH2 may serve core roles within the network and associate with one another during the pathogenesis of ESCC. These results indicate that a high expression of miR-25 promotes the invasion and metastasis of ESCC cells, while the influence of low expression of miR-25 differs with cells with different degrees of differentiation. Invasion and metastasis are not effected in cells with poor differentiation, while they were decreased in well differentiated cells. Furthermore, PTEN, TP53, MDM2, E2F1, PRMT5, MCM2, RB1, CDKN1A, SHAD7 and EZH2 may be targeted by the miR-106b-25 cluster, and act together to regulate the development of ESCC.

Beclin 1 expression is associated with the occurrence and development of esophageal squamous cell carcinoma.

Beclin 1 has a central role in the regulation of autophagy, differentiation, apoptosis resistance, tumorigenesis and cancer progression. The role of Beclin 1 in the development of esophageal squamous cell carcinoma (ESCC) and its subsequent progression is not fully characterized. In the present study, the role of Beclin 1 and autophagy in ESCC was evaluated. The expression of Beclin 1 mRNA and protein levels in human ESCC tumor and adjacent normal esophageal tissue was measured. Beclin 1 mRNA and protein were significantly lower in tumor tissue than in normal esophageal tissue (P<0.05). Cells of the less differentiated esophageal tumors expressed lower Beclin 1 mRNA and protein (P<0.05). Tumors from patients in early clinical stages (I/II) exhibited significantly higher Beclin 1 mRNA and protein expression levels than patients with tumors in mid-to-late stages (III/IV; P<0.05). Tumors from patients with lymph node metastasis exhibited significantly lower Beclin 1 mRNA and protein expression levels compared with tumors from patients without lymph node involvement (P<0.05). Beclin 1 downregulation was demonstrated to significantly upregulate invasion by ESCC EC9706 cells (P<0.01), and downregulate the number of acidic vesicular organelles, a process associated with autophagy. These results suggest that the expression of Beclin 1 is associated with the occurrence and development of ESCC. Measuring the Beclin 1 expression of tumors from patient may improve the understanding of the prognosis of patients with ESCC.

Expression of microRNA-935 in esophageal squamous cell carcinoma tissues and cells and its effects on cell proliferation and invasion

Objective To investigate the expression of microRNA (miRNA, miR)-935 in esophageal squamous cell carcinoma (ESCC) tissues and cells and explore its effects on proliferation and invasion ability of ESCC cells. Methods Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was employed to detect the expression of miR-935 in ESCC tissues and cells as well as normal esophageal tissues and cells. MiR-935 mimic, miR-935 inhibitor and negative control miRNA were utilized to transfect ESCC EC1 cells, and FQ-PCR was utilized to detect the expression of miR-935 48 h after transfection. Furthermore, cell proliferation and invasion ability was analyzed by cell counting kit-8 (CCK-8) and Transwell chamber after transfection. Results Expression of miR-935 in ESCC tissues and cells was significantly higher than that in normal esophageal tissues and cells, and the differences were statically significant (t=8.349, P=0.000). In addition, expression of miR-935 in metastatic ESCC patients were evidently higher than that in non-metastatic ESCC patients, and the differences were statically significant (t=4.264, P=0.000). MiR-935 mimic markedly upregulated the expression level of miR-935 in EC1 cells, whereas miR-935 inhibitor significantly downregulated the expression level of miR-935 in EC1 cells. Further investigation revealed that miR-935 upregulation promoted the proliferation and invasion ability of EC1 cells, conversely, miR-935 downregulation significantly inhibited the proliferation and invasion ability of EC1 cells. Conclusion MiR-935 plays an essential role in the occurrence and development of ESCC, and thus may be an underlying molecular target for the patients with ESCC. Key words: MicroRNA-935; Esophageal squamous cell carcinoma; Cell proliferation; Invasion; Metastasis

Abstract 1901: DNMT1 is involved in esophageal squamous cell carcinoma (ESCC) and self-renewal ability of ESCC-cancer stem cells

Abstract DNA methylation mediated by DNA methyltransferase 1 (DNMT1) plays an important role in carcinogenesis and self-renewal ability of cancer stem cells (CSCs). However, the function of DNMT1 in esophageal squamous cell carcinoma (ESCC) carcinogenesis, especially self-renewal ability of ESCC-CSCs remains unclear. In this study, we found a high expression of DNMT1 in both side population (SP) cells and sphere formation cells that represented as substitutes for CSCs in KYSE150 and EC109 ESCC cell lines. In addition, the expression of DNMT1 was decreased during the differentiation from SP to None-SP (NSP) in these ESCC cells. These results suggested that DNMT1 might have a role in regulating self-renewal and/or differentiation of ESCC-CSCs. To further investigate DNMT1 in ESCC carcinogenesis and self-renewal ability of ESCC-CSCs, we silenced the expression of DNMT1 in KYSE150 and EC109 ESCC cells using lentivirus-mediated RNA interference (RNAi). Our results showed that ablation of DNMT1 expression in KYSE150 and EC109 ESCC cells resulted in decreased their CSCs by SP analysis and sphere formation assay. Meanwhile, ablation of DNMT1 expression inhibited malignant phenotypes in KYSE150 and EC109 cells, including cell proliferation, colony formation, migration and drug resistance abilities. Treatment of 5-aza-2'-deoxycytidine (5-aza-dC), a DNMT inhibitor that led to the degradation of DNMT1 protein by proteasome, revealed that numbers of CSCs and the malignant phenotypes of KYSE150 and EC109 ESCC cells were refrained significantly, including a dramatic inhibition of self-renewal ability of these ESCC-CSCs. Thus, our results indicated that DNMT1 was involved in ESCC carcinogenesis, especially in the maintenance of ESCC-CSCs, suggesting that DNMT1 could be a potential target for ESCC, especially ESCC-CSCs, therapy. Citation Format: Ying Teng, Xiying Yu, Hui Yuan, Liping Guo, Wei Jiang, Shih-Hsin Lu. DNMT1 is involved in esophageal squamous cell carcinoma (ESCC) and self-renewal ability of ESCC-cancer stem cells [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 1901. doi:10.1158/1538-7445.AM2017-1901

Synthesis and anti-proliferative activity evaluation of novel benzo[d][1,3] dioxoles-fused 1,4-thiazepines

Benzo[d][1,3]dioxoles 1,4-thiazepines remarkable antitumor activities, benzo[d][1,3]dioxoles-fused 1,4-thiazepines, which combine two biologically active heterocyclic cores, are expected to be of pharmacological interest, We therefore envisaged that integrating 1,4-thiazepine and benzo[d][1,3]dioxole moieties in one molecular platform could potentially produce novel compounds with significant synergistic antitumor properties. A series of novel benzo[d][1,3]dioxoles-fused 1,4-thiazepines, designed via molecular hybridization approach, were synthesized in very good yields using one-pot condensation of 3,4-methylenedioxyaniline, aldehydes, and α-mercaptocarboxylic acids under solvent-free condition. The anti-proliferative activities of all the synthesized compounds were assessed on two different human cancer cell lines (Esophageal squamous cell carcinoma Ec9706 and Eca109), and the results showed that compound 4e showed the best anti-tumor activity with IC50 values of 8.23 μM and 16.22 μM against Ec9706 and Eca109 cell lines, respectively, which was 2–3 times more potent than 5-Fluorouracil (IC50 = 23.26 μM and 30.25 μM against Ec9706 and Eca109 respectively). These novel benzo[d][1,3]dioxoles fused with bioactive heterocyclic skeletons may find their pharmaceutical applications after further investigations.

HDAC2 regulates cell proliferation, cell cycle progression and cell apoptosis in esophageal squamous cell carcinoma EC9706 cells.

Increasing evidence has demonstrated that histone deacetylase 2 (HDAC2) participates in the regulation of a variety of biological processes in numerous tumors. However, the potential role of HDAC2 in the development and progression of esophageal squamous cell carcinoma (ESCC) remains elusive. Immunohistochemistry was utilized to detect the expression of HDAC2, Cell Counting Kit-8 was used to determine the cell proliferation, and flow cytometry was employed to investigate cell cycle and cell apoptosis. Finally, western blotting was employed to detect the protein expression of cyclin D1, p21, B cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax). The present study found that expression of HDAC2 protein in ESCC tissues was significantly increased compared with atypical hyperplasia tissues and normal esophageal mucosa (P<0.001). The expression of HDAC2 was not associated with the age or gender of patients (P>0.05), but was closely associated with the histological grade, invasion depth, tumor-node-metastasis stage and lymph node metastasis, respectively (all P<0.001). HDAC2 small interfering RNA effectively downregulated the expression of HDAC2 protein in ESCC EC9706 cells. Downregulation of HDAC2 expression evidently inhibited cell proliferation, arrested cell cycle at the G0/G1 phase and induced cell apoptosis in ESCC EC9706 cells, coupled with increased expression of p21 and Bax proteins and decreased expression of cyclin D1 and Bcl-2 proteins. Overall, the present findings suggest that HDAC2 may play an important role in the development and progression of ESCC and be considered as a novel molecular target for the treatment of ESCC.

Inhibition of autophagy promotes cell apoptosis induced by the proteasome inhibitor MG-132 in human esophageal squamous cell carcinoma EC9706 cells.

Lysosome-dependent macroautophagy, also termed autophagy, and the ubiquitin-proteasome system and are the primary intracellular pathways involved in protein degradation. Previous studies have demonstrated that proteasome inhibitors are able to inhibit tumor growth and activate autophagy. The present study investigated the effect of the proteasome inhibitor MG-132 on cellular proliferation using a cell counting kit 8 assay, and the effect of the agent on apoptosis and autophagy was assessed using flow cytometry and monodansylcadaverine, respectively. Western blot analysis was used to investigate protein changes during the course of treatment. It was revealed that MG-132 inhibited cell proliferation, activated autophagy and induced cell death in EC9706 cells. Autophagy was activated through the class III PI3K pathway, and the expression of the Beclin-1 protein was determined to be significantly upregulated. However, the autophagy inhibitor 3-methyladenine (3-MA) inhibited the expression of the autophagy-associated protein Beclin-1 and reduced the accumulation of autophagic vacuoles induced by MG-132. MG-132-induced apoptosis was enhanced by the autophagy inhibitor 3-MA, which may be a result of caspase-3 activation in the EC9706 cells. These findings suggest that inhibition of the proteasome can induce autophagy in human ESCC cells, and also increase cell death. This indicates that proteasome inhibitors may be potential novel anti-cancer agents for the adjuvant treatment of esophageal squamous cell carcinoma.

Functional analysis of the mRNA profile of neutrophil gelatinase‑associated lipocalin overexpression in esophageal squamous cell carcinoma using multiple bioinformatic tools.

Neutrophil gelatinase-associated lipocalin (NGAL) is a member of the lipocalin superfamily; dysregulated expression of NGAL has been observed in several benign and malignant diseases. In the present study, differentially expressed genes, in comparison with those of control cells, in the mRNA expression profile of EC109 esophageal squamous cell carcinoma (ESCC) cells following NGAL overexpression were analyzed by multiple bioinformatic tools for a comprehensive understanding. A total of 29 gene ontology (GO) terms associated with immune function, chromatin structure and gene transcription were identified among the differentially expressed genes (DEGs) in NGAL overexpressing cells. In addition to the detected GO categories, the results from the functional annotation chart revealed that the differentially expressed genes were also associated with 101 functional annotation category terms. A total of 59 subpathways associated locally with the differentially expressed genes were identified by subpathway analysis, a markedly greater total that detected by traditional pathway enrichment analysis only. Promoter analysis indicated that the potential transcription factors Snail, deltaEF1, Mycn, Arnt, MNB1A, PBF, E74A, Ubx, SPI1 and GATA2 were unique to the downregulated DEG promoters, while bZIP910, ZNF42 and SOX9 were unique for the upregulated DEG promoters. In conclusion, the understanding of the role of NGAL overexpression in ESCC has been improved through the present bioinformatic analysis.

Ethanol extract and isolated constituents from artemisia dracunculus inhibit esophageal squamous cell carcinoma and induce apoptotic cell death.

The objective of the present study was to examine the antitumor efficacy of the ethanol extract from Artemisia dracunculus as well as the compounds isolated from it on cultured EC‑109 esophageal squamous cell carcinoma (ESCC) cells. Apoptotic activities of the compounds were also studied using flow cytometry. EC‑109 esophageal cancer cells were treated with varying concentrations of compounds 1-7 isolated from the plant as well as the ethanol extract of Artemisia dracunculus. The cytotoxicity was evaluated by MTT assay and the apoptotic studies of the compounds were determined using flow-cytometry. Effect on mitochondrial membrane potential loss ΛΨ m induced by compounds 2 and 4 was also studied in these cells. Bioassay-guided fractionation of the ethanol extract from the shoot and root parts of Artemisia dracunculus led to the isolation of 7-methoxycoumarin (1), scopoletin (2), dracumerin (3), sakuranetin (4), elimicin (5), davidigenin (6) and 6-methoxycapillarisin (7). All the compounds as well as the extract showed mild to potent cell proliferation inhibitory activities against the esophageal cell line. Sakuranetin and 6-methoxycapillarisin were found to have the most potent effects in inhibiting the cell proliferation. The 2 potent compounds, sakuranetin and 6-methoxycapillarisin were evaluated for their effects on cell cycle phase distribution (DNA damage) as well as their effects on mitochondrial membrane potential loss ΛΨ m. Both compounds induced DNA damage as well as mitochondrial membrane potential loss in esophageal cancer cells. The study suggests that compounds, Sakuranetin and 6-methoxycapillarisin isolated from Artemisia dracunculus possess potent anticancer effects by inducing DNA damage in these cells.

Effect of downregulation of Tiam1 by siRNA on esophageal squamous cell carcinoma EC9706 cells

To explore the effect of downregulation of Tiam1 by siRNA on the esophageal squamous cell carcinoma (ESCC) EC9706 cells, and provide theoretical basis for gene therapy of ESCC using Tiam1 as a molecular target. Tiam1 siRNA was transfected into EC9706 cells, and expression changes of Tiam1 mRNA and protein after transfection were detected by quantitative real-time PCR and Western blotting. Cell proliferation was analyzed using CCK-8 kit. Cell cycle and apoptosis of the EC9706 cells were assessed by flow cytometry. Cell cycle-related proteins and cell apoptosis-associated proteins were analyzed by Western blotting. Compared with the untreated group and control siRNA group, the relative expression levels of Tiam1 mRNA (1.00 and 0.11 ± 0.02) were not significantly different (P > 0.05). The relative expression levels of Tiam1 mRNA in the Tiam1 siRNA group at 24, 48 and 72 h after transfection were 0.30 ± 0.04, 0.09 ± 0.01 and 0.09 ± 0.006, respectively, significantly lower than that of the untreated group (P < 0.05 for all). The expression level of Tiam1 protein at 24 h after Tiam1 siRNA transfection in the EC9706 cells was 0.11 ± 0.02, significantly lower than that in the un-treated group (0.44 ± 0.05) and control siRNA group (0.44 ± 0.04, P < 0.05 for all). The percentages of G0/G1 cells in the Tiam1 siRNA group, untreated group and control siRNA group were (54.48 ± 2.14)%, (40.69 ± 1.85)% and (41.78 ± 1.31)%, respectively (P < 0.01). The percentages of S phase cells in the Tiam1 siRNA group, untreated group and control siRNA group were (27.18 ± 1.65)%, (32.32 ± 1.15)% and (30.35 ± 1.09)%, respectively (P < 0.01). The expression levels of cyclin D1 protein in the untreated group, control siRNA group and Tiam1 siRNA group were 0.43 ± 0.02, 0.41 ± 0.01 and 0.11 ± 0.02, respectively (P < 0.05). The expression levels of p27 protein in the untreated group, control siRNA group and Tiam1 siRNA group were 0.10 ± 0.01, 0.09 ± 0.02 and 0.20 ± 0.02, respectively (P < 0.05). The ratios of early apoptotic cells in the untreated group, control siRNA group and Tiam1 siRNA group were (10 ± 0.9)%, (10 ± 0.5)% and (27 ± 0.7)%, respectively (P < 0.01). The expression levels of Mcl-1 protein in EC9706 cells of untreated group, control siRNA group and Tiam1 siRNA group were 0.47 ± 0.12, 0.48 ± 0.13 and 0.16 ± 0.02, respectively (P < 0.05). The expression levels of Bcl-2 protein in EC9706 cells of the untreated group, control siRNA group and Tiam1 siRNA group were 0.49 ± 0.08, 0.50 ± 0.05 and 0.04 ± 0.03, respectively (P < 0.05). The caspase-3 activities in the untreated group, control siRNA group and Tiam1 siRNA group were 2.3 ± 0.09, 2.3 ± 0.10 and 16.0 ± 1.50, respectively; and that of caspase-9 were 2.3 ± 0.08, 2.3 ± 0.11 and 14.5 ± 0.9, respectively (P < 0.05 for all). Tiam1 siRNA can significantly inhibit the proliferation of esophageal cancer EC9706 cells, induce cell cycle arrest and cell apoptosis. These effects are related to the regulation of the expressions of cell cycle-related genes (cyclin D1 and p27) and cell apoptosis-related genes (Mcl-1, Bcl-1, caspase-3 and caspase-9) by Tiam1 siRNA.

Insulin enhances apoptosis induced by cisplatin in human esophageal squamous cell carcinoma EC9706 cells related to inhibition of autophagy

Background Chemoresistance is common among patients with esophageal squamous cell carcinoma (ESCC). We investigated the effect and mechanism of insulin on enhancing anticancer functions of cisplatin in human esophageal cancer cell line EC9706. Methods The viability of EC9706 cells exposed to cisplatin was assessed using MTT assay. The times T1, when the number of living cells reached a plateau and T2, when the number of living cells reached a new plateau after the addition of insulin were found. T1 and T2 plateau cells were stained by Annexin V-FITC/PI and monodansylcadaverin (MDC). Fluorescent microscopy was used to observe the expression of apoptosis and autophagy intuitively. Apoptotic ratio and fluorescent intensity were analysed by flow cytometry (FCM) quantitatively. Western blotting analysis was used to estimate the protein expression levels of AKT, mTOR, PI3K, PTEN, autophage related indicator LC3-II and autophage related protein Beclin1 changes that occurred in the course of treatment. Results A larger number of typical autophagosomes were detected in EC9706 cells exposed to cisplatin. Insulin can increase the apoptosis induced by cisplatin. Apoptotic ratio of T1 plateau cells ((32.6±4.3)%) is significantly less than T2 plateau ((47.5±5.6)%). MDC fluorescent intensity at T1 plateau (104.9±13.2) was significantly higher than intensity at T2 plateau (82.6±10.3). After cotreatment with insulin, the expression level of LC3-II, Beclin1 and PTEN in T2 plateau cells were significantly downregulated, but AKT, mTOR and PI3K expressions significantly upregulated compared with T1 plateau. Conclusions Insulin could enhance cisplatin-induced apoptosis in human esophageal squamous cell carcinoma EC9706 cells related to inhibition of autophagy. The activation of PI3K/Akt/mTOR signaling pathway induced by insulin resulted in the suppression of autophagy in EC9706 cells, which may be attributed to the anticancer effects of cisplatin.

Effects of S100A7 on the proliferation and invasiveness of esophageal squamous cell carcinoma EC9706 cells and possible mechanisms

To explore the expression of S100A7 in esophageal squamous cell carcinoma (ESCC) and examine the effect of S100A7 down-regulated expression mediated via RNA interfering on cell proliferation and invasion. The expressions of S100A7 mRNA and protein were analyzed by in situ hybridization and immunohistochemistry.S100A7 siRNA was transfected into ESCC cell line EC9706 and the expressions of S100A7 mRNA and protein were detected by real-time polymerase chain reaction (PCR) and Western blot.Furthermore, the effects of down-regulated of S100A7 on cell proliferation and invasion were examine by Cell Counting Kit-8 (CCK-8) and Boyden chamber respectively.Finally the effect of down-regulated expression of S100A7 on matrix metalloproteinase-2 (MMP-2) protein was analyzed by Western blot. Positive expression ratios of S100A7 mRNA and protein expressions in ESCC tissues (72.0% and 69.3%) were significantly higher than those in normal esophageal tissues (10.7% and 8.0%) (χ² = 58.174 and 59.483, both P = 0.000). The expressions of S100A7 mRNA and protein in S100A7 siRNA group were markedly lower than those in untreated group (mRNA: 0.235 ± 0.056 vs 1, protein: 0.119 ± 0.025 vs 0.518 ± 0.129, both P < 0.05). Additionally, the down-regulated expression of S100A7 resulted in the proliferation inhibition and the decreased invasiveness in EC9706 cells. And it was coupled with a down-regulation of MMP-2 protein. S100A7 may play an essential role in the occurrence and development of ESCC and the decreased invasiveness of EC9706 cells mediated by the down-regulated expression of S100A7 may be closely associated with the down-regulation of MMP-2 protein.

Effects of downregulation of SIRT3 expression on proliferation and apoptosis in esophageal squamous cell carcinoma EC9706 cells and its molecular mechanisms.

To investigate the effect of downregulation of SIRT3 expression on cell proliferation and invasion in esophageal squamous cell carcinoma (ESCC) EC9706 cells, and to explore its possible molecular mechanisms, we transfected siRNA targeting SIRT3 into EC9706 cells, and then divided cells into three groups: untreated, control siRNA and SIRT3 siRNA groups. The expression levels of SIRT3 protein were detected in different groups by western blotting. The effect of SIRT3 siRNA on cell proliferation was investigated using the CCK-8 kit. The changes of cell apoptosis were examined by flow cytometry. Finally, the expression levels of cell proliferation and apoptosis related proteins such as p21, Bcl-2 and Bax were determined by western blotting. SIRT3 siRNA effectively down-regulated the expression of SIRT3 protein in EC9706 cells, and the reduced expression of SIRT3 significantly inhibited cell proliferation and induced cell apoptosis. Most notably, the SIRT3 depletion markedly increased the expressions of p21 and Bax proteins but reduced Bcl-2 protein expression. The proliferation inhibition and apoptosis of EC9706 cells mediated by SIRT3 downregulation may be closely associated with the expression levels of p21, Bcl-2 and Bax proteins.

Baicalein induces apoptosis in esophageal squamous cell carcinoma cells through modulation of the PI3K/Akt pathway

Baicalein, a flavone present in Scutellaria baicalensis Georgi, has been demonstrated to possess antitumor activity in a variety of cancer cells in vitro. However, its effects on the growth inhibition and induction of apoptosis in human esophageal carcinoma cells remain unclear. The aims of this study were to determine whether cultured EC-109 esophageal squamous cell carcinoma (ESCC) cells undergo apoptosis when treated with baicalein and to investigate the underlying mechanisms in vitro. Cell growth was measured using MTT and plate colony formation assays. Induction of apoptosis was examined using Hoechst 33258 staining, flow cytometry analysis and a DNA fragmentation assay. The mechanisms underlying the observed growth suppression were examined using western blot analysis. The results demonstrated that treatment of EC-109 cells with baicalein for 48 h markedly decreased the rate of cell viability. Colony formation was almost fully suppressed at 40 μM baicalein. EC-109 cells underwent apoptosis in response to baicalein treatment, demonstrated by an increase in the percentage of cells stainable with Hoechst 33258 and Annexin V-FITC/PI, increased DNA fragmentation and activation of the intrinsic (mitochondrial) pathway for cell death. The latter was characterized by increased expression of the cleaved forms of caspase-9 and -3, and poly (ADP-ribose) polymerase (PARP). Additionally, baicalein was found to downregulate anti-apoptotic components and upregulate apoptotic components of the PI3K/Akt pathway. In conclusion, baicalein induces apoptosis in EC-109 cells through modulation of the PI3K/Akt pathway, thus providing further understanding of the molecular mechanisms of baicalein action in esophageal carcinoma. Therefore, the present study revealed that baicalein significantly inhibits growth and induces apoptosis in EC-109 human ESCC cells in vitro.

RETRACTED ARTICLE: HAX-1 Promotes the Chemoresistance, Invasion, and Tumorigenicity of Esophageal Squamous Carcinoma Cells

HAX-1 is an anti-apoptotic factor and regulates the expression of DNA pol β. Interestingly, DNA polymerase pol β is overexpressed in esophageal squamous cell carcinoma (ESCC). However, the functional role of HAX-1 in ESCC remains unclear. To investigate the role of HAX-1 in chemoresistance, invasion, and tumorigenicity of ESCC. Lentivirus-mediated overexpression or knockdown of HAX-1 was employed to establish ESCC EC9706 cell lines that expressed HAX-1 at different levels. The biological behaviors of these engineered cells were characterized in vitro and in vivo using a xenograft nude mice model. In addition, HAX-1 and pol β expression in the tumor tissues was detected by RT-PCR and immunohistochemistry. HAX-1 overexpression promoted cell proliferation and resistance against cisplatin, increased cell invasion and suppressed apoptosis along with increased pol β expression. Conversely, HAX-1 knockdown inhibited the malignant phenotypes of EC9706 cells. The xenograft nude mice model demonstrated that HAX-1 overexpression or depletion led to increased or decreased tumor growth in vivo, respectively. Furthermore, a positive correlation of HAX-1 and pol β expression in the tumor tissues was observed. HAX-1 promotes the proliferation, chemoresistance, invasion, and tumorigenicity of ESCC, and this is correlated with increased poly β expression. HAX-1 may represent a potential target to overcome the resistance and metastasis of ESCC.

Expression of IGF-1R in esophageal squamous cell carcinoma and the effect of its silencing by siRNA on the proliferation of esophageal cancer EC9706 cells in vitro

To explore the correlation of IGF-1R expression with clinical features of esophageal squamous cell carcinoma (ESCC) and to investigate the effect of silencing IGF-1R by siRNA on the proliferation of esophageal cancer cell line EC9706 cells. Immunohistochemistry was used to detect the expresion of IGF-1R in 80 specimens of ESCC and 18 specimens of normal esophageal mucosa. IGF-1R siRNA was transfected into esophageal squamous cell carcinoma EC9706 cells, and the effect of RNAi was assessed by Western blot. The proliferation of EC9706 cells was determined by drawing growth curve, MTT assay and plate colony-forming assay. The total and strong positive rates of IGF-1R expression were 86.3% and 51.3% in ESCC, and 61.1% and 11.1% in normal esophageal epithelium, respectively. The total and strong positive rates of IGF-1R expression in patients with lymph node metastasis were 94.4% and 74.1%, significantly higher than 69.2% and 3.9%, respectively, in those without lymph node metastasis (P<0.01). A significantly higher IGF-1R expression was associated with lower histological grade (P<0.05). The total and strong rates of IGF-1R expression in 39 patients of stages III and IV were 97.4% and 71.8% , significantly higher than the 75.6% and 31.7%, respectively, in 41 cases of stages I and II (P<0.01). IGF-1R RNAi significantly inhibited IGF-1R expression and the growth of EC9706 cells. The clone formation rate of RNAi-IGF-1R transfected cells was 19.1%, significantly lower than that of 52.3% in non-transfected cells and 49.0% in empty vector-transfected EC9706 cells (P<0.05). The overexpression of IGF-1R is colerated with lymph node metastasis, differentiation and clinical stage. Down-regulation of IGF-1R can inhibit the proliferation of esophageal cancer EC9706 cells in vitro.