EC9706 Cells Research Articles

TKTL1 expression and its downregulation is implicated in cell proliferation inhibition and cell cycle arrest in esophageal squamous cell carcinoma.

It is well known that tumor cells mainly depend on the nonoxidative pathway of the pentose phosphate pathway (PPP), and transketolase-like 1 (TKTL1), a kind of crucial metabolism enzyme, participates in the regulation of PPP; notably, overwhelming evidence has demonstrated that TKTL1 plays pivotal roles in the development and progression of multiple tumors. However, there were no reports about the role of TKTL1 in esophageal squamous cell carcinoma (ESCC). Here, we investigated TKTL1 expression and preliminarily elucidated its underlying biological functions in ESCC. We found that TKTL1 exhibited the high expression in ESCC tissues and cells, and the survival rate of patients with negative TKTL1 expression was significantly higher than that of patients with positive TKTL1 staining (P < 0.05). Additionally, significant correlations of TKTL1 expression with histologic grade, clinical stage, and lymph node metastasis were found (P < 0.05). Subsequently, TKTL1 small interfering RNA (siRNA) significantly reduced TKTL1 messenger RNA (mRNA), and protein levels companied with the marked reduce of total transketolase activity but did not affect TKT and TKTL1 mRNA level. More importantly, TKTL1 siRNA obviously induced cell cycle arrest in G0/G1 phase and suppressed cell proliferation in vitro and in vivo coupled with the reduced cyclin D1 and cdk4 levels as well as decrease of Ki-67 proliferation index in EC1 cells. Taken altogether, our results suggest that TKTL1 as a key prognostic factor may be a novel target for therapy of the patients with ESCC.

Regulation of α-tocopherol on NFκB and Nrf2 signaling pathway at early stage of N-nitrosomethylbenzylamine⁃induced human esophageal cell carcinogenesis

To investigate the regulation of α-Tocopherol on NFκB and Nrf2 signaling pathway at early stage of N-nitrosomethylbenzylamine (NMBzA)-induced human esophageal carcinogenesis. Human normal esophageal HET-1A cells were treated with NMBzA at 50 µmol/L, 100 µmol/L for 24 h to intimate the initiation of esophageal carcinogenesis. For intervention groups, HET-1A cells were pre-treated with α-T at 25, 50, 100 µmol/L for 3 h and then co-treated with NMBzA (100 µmol/L) for 24 h. In comparison with HET-1A cells, human esophageal cancer EC109 cells were treated with α-T at corresponding concentrations. Cells treated with 0.1% DMSO were used as negative control. Immunofluorence staining was used for the determination of distribution and activation of NFκB p65 and Nrf2 in the cell. Real time PCR and Western blot were used to determine the expression levels of target genes including cyclinD1, KI67, proliferating cell nuclear antigen (PCNA), cyclo-oxygen-ase 2 (COX2), 5LOX, HO-1, NQO1 and GCLC. Flow cytometry was utilized to analyze the reactive oxygen species contents in the cells. As compared to the control group (1.00 ± 0.08), the expression of CyclinD1 (2.99 ± 0.15), KI67 (2.35 ± 0.38) and PCNA (2.46 ± 0.25) in HET-1A were all markedly increased by NMBzA treatment (F values were 97.23, 65.28, 34.62, P < 0.001). Also, the proportion of cells with nucleus translocation of NFκB p65 (71.0%, 98/138) or Nrf2 (36.3%, 49/135) were significantly increased (χ² values were 194.71, 133.72, P < 0.001), and the expression of COX2 (3.22 ± 0.17), 5LOX (2.87 ± 0.12) as well as HO-1 (1.87 ± 0.22), NQO1 (2.14 ± 0.08), GCLC (2.63 ± 0.41) at protein levels were elevated (F values were 72.35, 43.87, 69.23, 71.34, 85.79, P values were 0.013, 0.015, 0.010, 0.011, 0.002). Under the treatment with 50 µmol/L α-T, comparing with the control group(59.1%,65/110),the nuclear translocation of NFκB p65 (77.7%, 8/104) was clearly inhibited (χ² = 148.1, P < 0.001), and protein expression levels of COX2 (0.74 ± 0.19) and 5LOX (0.42 ± 0.13) were decreased (F values were 56.31, 73.25, P values were 0.003, 0.001). However, no changes on Nrf2 signaling pathway were observed; α-T showed little impact on NFκB or Nrf2 pathway in EC109 cells. At the early stage of NMBz-induced esophageal cancer, α-T could block the initiation of carcinogenesis through suppressing the activation of NFκB signaling pathway. It might be the major mechanism by which α-T is potentially chemopreventive to esophageal cancer. During the progression of esophageal cancer, the cells may acquire the adaptive functions to accommodate oxidative stress via activating Nrf2 pathway.

Curcumin inhibits Ec109 cell growth via an AMPK-mediated metabolic switch.

Glycolytic enzymes are always greatly increased in cancer cells. Whether metabolic reprogramming is involved in curcumin-mediated inhibition of cancer cell growth is unknown. In this study, cell viability was assayed with MTS analysis; cell cycle was measured with flow cytometry analysis. RT-PCR and western blotting were used to analyse the mRNA and protein expression, respectively. Here we demonstrated that curcumin inhibited cancer cell growth, especially for Ec109 cells. Curcumin induced cell cycle arrest at G2/M phase. Curcumin caused a significant down-regulation of glycolytic enzymes expressions in a dose-dependent manner. Our results further indicated that the AMPK was required for curcumin-mediated down-regulation of glycolytic enzymes. AMPK-mediated down-regulation of glycolytic enzymes blocked Ec109 cell growth. Taken together, our results revealed that the AMPK-mediated metabolic switch plays an important role in esophageal cancer cell growth.

Metadherin is required for the proliferation, migration, and invasion of esophageal squamous cell carcinoma and its meta-analysis

Metadherin (MTDH) was found to be highly expressed in various squamous cell carcinomas (SCCs); however, meta-analysis evaluating the association of MTDH in SCC has not been performed. The purpose of this study was to explore the biological functions of MTDH in esophageal squamous cell carcinoma (ESCC) and to meta-analyze the association between MTDH and SCC. Immunohistochemistry was performed to examine MTDH expression using an ESCC tissue array consisting of 86 ESCC and 78 paired normal adjacent tissues (NATs). MTDH was significantly overexpressed in ESCC tissues compared with NATs and was significantly associated with lymph node metastasis, differentiation, and prognosis. Knockdown of MTDH using an MTDH-short hairpin RNA plasmid caused cell cycle arrest at the G0/G1 phase and induced apoptosis of EC9706 cells. Knockdown of MTDH suppressed the proliferation, invasion, and migration of ESCC cells. Furthermore, meta-analysis revealed that overexpression of MTDH was significantly associated with the lymph node metastasis, advanced clinical stage, and T classification of tissues in SCC, suggesting that MTDH might be used as a potential therapeutic target in the lymph node metastasis of ESCC.

Abnormal Localization and Tumor Suppressor Function of Epithelial Tissue-Specific Transcription Factor ESE3 in Esophageal Squamous Cell Carcinoma.

Esophageal cancer is one of the most common malignant cancers worldwide. The molecular mechanism of esophageal squamous cell carcinoma (ESCC) is still poorly understood. ESE3 is a member of the Ets transcription family, which is only expressed in epithelial tissues and acts as a tumor suppressor gene in prostate cancer. Our study aim was to confirm whether ESE3 is involved in the carcinogenesis of ESCC. Immunohistochemical analysis revealed that ESE3 was mainly located in cell nuclei of normal tissues and the cytoplasm in ESCC tissues. Immunofluorescence and western blot analyses of the normal esophageal cell line HEEpiC and ESCC cell lines EC9706 TE-1, KYSE150, and KYSE410 confirmed these results. pEGFP-ESE3 and pcDNA3.1-V5/HisA-ESE3 plasmids were constructed for overexpression of ESE3 in EC9706 and KYSE150 cells. The stably transfected cells showed restoration of the nuclear localization of ESE3. EC9706 cells with re-localization of ESE3 to the nucleus showed inhibition of proliferation, colony formation, migration, and invasion. To explore the possible mechanism of the differences in localization of ESE3 in normal esophageal cells and ESCC cells, ESCC cell lines were treated with the nuclear export inhibitor leptomycin B, transcription inhibitor actinomycin D, PKC inhibitor sphinganine, P38 MAPK inhibitor SB202190, and CK II inhibitor TBCA. These reagents were chosen according to the well-known mechanisms of protein translocation. However, the localization of ESE3 was unchanged after these treatments. The sequence of ESE3 cDNA in ESCC cells was identical to the standard sequence of ESE3 in the NCBI Genebank database, indicating that there was no mutation in the coding region of ESE3 in ESCC. Taken together, our study suggests that ESE3 plays an important role in the carcinogenesis of ESCC through changes in subcellular localization and may act as a tumor suppressor gene in ESCC, although the mechanisms require further study.

Combining radiation with autophagy inhibition enhances suppression of tumor growth and angiogenesis in esophageal cancer.

Radiotherapy is an effective treatment for esophageal cancer; however, tumor resistance to radiation remains a major biological problem. The present study aimed to investigate whether inhibition of autophagy may decrease overall tumor resistance to radiation. The effects of the autophagy inhibitor 3-methyladenine (3-MA) on radiosensitivity were tested in the EC9706 human esophageal squamous cell carcinoma cell line by colony formation assay. Furthermore, the synergistic cytotoxic effects of 3-MA and radiation were assessed in a tumor xenograft model in nude mice. Mechanistic studies were performed using flow cytometry, immunohistochemistry and western blot analysis. The results of the present study demonstrated that radiation induced an accumulation of autophagosomes and 3-MA effectively inhibited radiation-induced autophagy. Inhibition of autophagy was shown to significantly increase the radiosensitivity of the tumors in vitro and in vivo. The enhancement ratio of sensitization in EC9706 cells was 1.76 when the cells were treated with 10 mM 3-MA, alongside ionizing radiation. In addition, autophagy inhibition increased apoptosis and reduced tumor cell proliferation. The combination of radiation and autophagy inhibition resulted in a significant reduction in tumor volume and vasculature in the murine model. The present study demonstrated in vitro and in vivo that radiation-induced autophagy has a protective effect against cell death, and inhibition of autophagy is able to enhance the radiosensitivity of esophageal squamous cell carcinoma.

Role of S100A4 in the epithelial-mesenchymal transition of esophageal squamous cell carcinoma and its molecular mechanism

To explore the role of S100A4 in the epithelial-mesenchymal transition (EMT) in esophageal squamous cell carcinoma and its possible molecular mechanism. Three chemically synthesized S100A4 siRNA sequences were transiently transfected into esophageal carcinoma EC9706 cells. EC9706 cells transfected with negative siRNA, lipofectamine 2000, and vacant EC9706 cells were used as control. Fluorescence quantitative RT-PCR and Western blot were used to detect the inhibition rate of S100A4 siRNA. S100A4 siRNA2 with the best inhibition rate was chosen to transiently transfect into EC9706 cells under the same conditions. The EC9706 cells transfected with negative siRNA, lipofectamine 2000 and vacant EC9706 cells were also used as control. Fluorescence quantitative RT-PCR and Western blot were used to detect the mRNA and protein expressions of E-cadherin, vimentin and snail. The morphology of EC9706 cells was observed under an inverted microscope. Boyden chamber and scratch test were used to detect the invasion and migration ability of EC9706 cells, and CCK8 assay was used to detect the proliferation ability of EC9706 cells. EC9706 cells transfected with S100A4 siRNA2 were further transfected with snail eukaryotic expression vector. The EC9706 cells transfected with S100A4 siRNA, EC9706 cells transfected with snail eukaryotic expression vector and vacant EC9706 cells were used as control. The above indexes of all the groups were observed, too. The S100A4 mRNA and protein expression levels of the S100A4 siRNA2 group were 0.417 ± 0.041 and 0.337 ± 0.039, the transmembrane cell number was 61.608 ± 8.937, the scratch healing distance was (0.216 ± 0.064) mm, the A value was 0.623 ± 0.084, the E-cadherin mRNA and protein levels were 0.619 ± 0.032 and 0.495 ± 0.034, the vimentin mRNA and protein levels were 0.514 ± 0.032 and 0.427 ± 0.028, the snail mRNA and protein levels were 0.573 ± 0.029 and 0.429 ± 0.041. These data were significantly different with the liposome group, the negative control group and the blank group (P < 0.05 for all). After the S100A4 siRNA2 treatment for 24 h, the appearance of EC9706 cells changed to epithelial cell morphology. The transmembrane cell number and the scratch healing distance of the S100A4 siRNA2+snail eukaryotic expression vector group were (69.382 ± 9.666) cells and (0.274 ± 0.029) mm, the A value was 0.823 ± 0.101, the snail mRNA and protein levels were 0.704 ± 0.037 and 0.625 ± 0.031, the vimentin mRNA and protein levels were 0.712 ± 0.046 and 0.609 ± 0.038, and these data were significantly higher than those of the Sl00A4 siRNA2 group (P < 0.05 for all). The E-cadherin mRNA and protein levels of the S100A4 siRNA2+eukaryotic expression vector group were 0.437 ± 0.038 and 0.381 ± 0.031, significantly lower than those of the S100A4 siRNA2 group (P < 0.05 for all). However, snail had no effect on the morphology of EC9706 cells. S100A4 may be involved in the EMT process of esophageal squamous-cell carcinoma by regulating the expression of snail and then plays a role in the invasion and metastasis of esophageal carcinoma.

MiR-889 promotes proliferation of esophageal squamous cell carcinomas through DAB2IP

MicroRNAs have been reported to play critical roles in various cancers, but there has been no study on the role of miR-889 in cancers. Here, we report that over-expression of miR-889 leads to rapid proliferation of EC109 and EC9706 cells in vitro and in vivo by inducing cells into S-phase. Using bioinformatics methods, DAB2IP was further confirmed to be a direct target of miR-889. In addition, the expression of DAB2IP, which was negatively correlated with that of miR-889, was significantly associated with clinicopathological features of ESCC patients. In conclusion, miR-889 is an important regulator in ESCC and both miR-889 and DAB2IP may serve as promising biomarkers and therapeutic targets in patients with ESCC.

Synthesis and antitumor activity of α-aminophosphonate derivatives containing thieno[2,3-d]pyrimidines

Abstract Two series of thieno[2,3-d]pyrimidine derivatives were designed and synthesized, in which bioactive α -aminophosphonate subunits were introduced at the N3 position through an N N bond connection. The in vitro cytotoxic activity of the novel compounds was tested against human esophageal carcinoma cells (EC109), human hepatocarcinoma cells (HepG2), human gastric carcinoma cells (MGC-803), respectively, by the MTT method. The evaluation results revealed that compounds 6mb , 6mf , 6mg , 6nd and 6nh exerted the most potent inhibition against HepG2, MGC-803 and EC109 cells, respectively. In particular, compound 6mg presented excellent inhibitory effect against HepG2 (91.2%) and MGC-803 (94.4%) cells.

Effects of silencing the ATP-binding cassette protein E1 gene by electroporation on the proliferation and migration of EC109 human esophageal cancer cells.

In the present study, the gene expression of ATP-binding cassette protein E1 (ABCE1) in the EC109 human esophageal cancer cell line was silenced using electroporation to examine the effect if the ABCE1 gene on the growth migration and cell cycle of cancer cells. The small interference (si)RNA sequence of ABCE1 was designed and synthesized to transfect the EC109 cells by electroporation. The mRNA and protein expression levels of ABCE1 were then detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The analysis of the cell cycle and apoptosis was performed using flow cytometry. The effect of silencing the ABCE1 gene on the proliferation, migration and invasive ability of the EC109 human esophageal cancer cells were assessed using a Cell counting kit-8 (CCK-8) and with proliferation, wound-healing and cell invasion assays. The mRNA and protein expression levels of ABCE1 were significantly lower in the experimental group compared with the control group (P<0.05). The apop-totic rate of the experimental group was markedly higher than the control group and blank group (P<0.01). The CCK-8 proliferation assay revealed that, compared with the control and blank groups, the proliferation of the EC109 cells in the experimental group was significantly inhibited (P<0.05). The wound healing assay revealed that the migration capacity of the cells in the experimental group was significantly decreased (P<0.05). The Transwell chamber assay demonstrated that the invasive ability of the EC109 cells in the experimental group was significantly decreased (P<0.01). These results revealed that ABCE1 is closely associated with cell proliferation, invasion and migration in esophageal cancer and silencing the ABCE1 gene by electroporation can significantly reduce the proliferation, invasion and migration capacity of EC109 cells in vitro.

Effect of anionic PEGylated polypeptide on gene transfection mediated by glycolipid conjugate micelles.

To improve the gene transfection efficiency mediated by chitosan-g-stearic acid (CS) micelles, poly(ethylene glycol)-b-poly(γ-glutamic acid) (PG) was incorporated into a CS-based gene delivery system. CS/PG/pDNA complexes were prepared by ionic interaction. CS and PEGylated CS (PCS) micelles were introduced to prepare binary complexes for use as controls. CS/PG/pDNA complexes possessed similar sizes and presented as irregular spheroids in shape. The incorporation of PG into CS/pDNA complexes did not affect the ability of CS to compact pDNA and also showed a protective effect against DNase I based degradation of pDNA. Importantly, PG could increase gene transfection efficiency, which was also affected by the mixing methods used for the preparation of CS/PG/pDNA ternary complexes. The transfection efficiencies mediated by CS/PG/pDNA complexes against HEK293 and EC-1 cells reached up to 40.8% and 11.6%, respectively, which were much higher than those of CS/pDNA complexes (1.3% and 4.0%) and PCS/pDNA complexes (0.8% and 2.4%). In addition, the incorporation of PG into CS/pDNA complexes significantly enhanced cellular uptake in HEK293 and EC-1 cells and, additionally, improved endosomal escape and intracellular vector unpacking. However, the incorporation of PG reduced the cellular uptake of CS/PG/pDNA complexes in macrophages (RAW264.7 cells). It was further demonstrated that, in addition to a nonspecific charge-mediated binding to cell membranes, a γ-PGA-specific receptor-mediated pathway was involved in the internalization of CS/PG/pDNA complexes. These results indicated that PG played multiple important roles in enhancing the transfection efficiency of CS/PG/pDNA complexes.

Esophageal cancer related gene 4 induced block of esophageal cancer cells in G1 phase by p53 pathway

Objective To investigate the tumor-suppressing function and mechanism of esophageal cancer related gene 4(ECRG4)in esophageal carcinoma. Methods The ECRG4 gene expression plasmid pcDNA3.1-ECRG4 was constructed, and the EC9706 cell line with ECRG4 gene stable transfection was achieved. The effect of ECRG4 gene over-expression on esophageal cancer cells proliferation in vitro and tumor growth in vivo was studied by cell growth curves and tumor formation in nude mice experiments. The cell cycle change was examined by flow cytometry. The cell cycle regulation genes p53 and p21 protein expression changes were detected by Western blotting. Results The esophageal cancer cells proliferation in vitro and tumor growth in vivo were decreased in ECRG4 over- expression group,and the in vivo tumor growth inhibition rate was 48.5%,as compared with empty plasmid control group(P<0.05).ECRG4 over- expression induced block of esophageal cancer cells in G1 phase as compared with empty plasmid control group(P< 0.05).The p53 and p21 protein expression was increased in ECRG4 over- expression group as compared with empty plasmid control group(P<0.05). Conclusion ECRG4 induced block of esophageal cancer cells in G1 phase by p53 pathway. Key words: Esophageal cancer related gene 4; p53; p21; Esophageal cancer

Micro ribonucleic acid-93 promotes proliferation and migration of esophageal squamous cell carcinoma by targeting disabled 2.

BackgroundAccumulated evidence has revealed that the dysregulation of micro ribonucleic acids (miRNAs) may contribute to esophageal squamous cell carcinoma (ESCC). MiR-93, which is a member of the miRNA cluster miR-106b∼25, has been widely studied for its tumor promoting effect on different types of cancers. However, our knowledge of miR-93 function in ESCC remains unclear.MethodsThe expression levels of miR-93 in ESCC and the adjacent non-tumor tissues were measured by real-time polymerase chain reaction. Cell counting kit-8, flow cytometry, and 5-ethynyl-2′-deoxyuridine incorporation and transwell migration assays were employed to explore the effects of miR-93 on proliferation and migration capabilities in EC109 cells. To determine the possible target gene of miR-93, cell transfection, Western blot analysis and luciferase reporter gene assays were performed.ResultsA significant upregulation of miR-93 expression in ESCC tissues was determined, combined with a downregulation of the predicted target gene, disabled 2 (DAB2). The introduction of miR-93 significantly promotes cell proliferation, cell cycle progression, and the metastatic capability of EC109 cells. By cell transfection and luciferase reporter assay, DAB2 was confirmed as a direct target of miR-93. In addition, the knockdown of DAB2 by small interfering RNA displayed a consentaneous phenocopy with miR-93 overexpression in EC109 cells.ConclusionOur results indicate that miR-93 acts as a tumor promoter in ESCC, and its promotion effects on ESCC cell proliferation and migration depend largely upon DAB2 suppression.

MiRNA-1207-5p is associated with cancer progression by targeting stomatin-like protein 2 in esophageal carcinoma.

Newly discovered intrinsic regulators, the miRNAs regulate gene expression by binding to the 3'-untranslated regions of the genome. Accumulating studies have indicated that miRNAs are aberrantly expressed in various human cancers. We found that miRNA-1207-5p (miR‑1207-5p) was markedly downregulated in esophageal carcinoma (EC) tissues, and was correlated with EC differentiation, pathological stage and lymph node metastasis. Rates of apoptosis were increased and cell invasion ability was decreased in EC9706 and EC-1 cells transfected with a miR‑1207-5p mimic. Stomatin-like protein 2 (STOML-2) was predicted to be a potential target of miR‑1207-5p by bioinformatics analysis and this was confirmed by luciferase assay and western blotting. Our study showed that STOML-2 was negatively regulated by miR‑1207-5p. Furthermore, overexpression of STOML-2 abolished the miR‑1207-5p anti-invasion function. Based on these results, we proposed that miR‑1207-5p might act as a potential therapeutic target in the treatment of EC.

Indomethacin induces apoptosis in the EC109 esophageal cancer cell line by releasing second mitochondria-derived activator of caspase and activating caspase-3.

The use of non‑steroidal anti‑inflammatory drugs (NSAIDs) has been associated with a reduced risk of various types of cancer, including esophageal cancer. However, the mechanisms underlying the antineoplastic effects of NSAIDs in esophageal cancer remain to be elucidated. In the present study, a significant inhibition in cell viability was observed in the EC109 cells following treatment with different concentrations of indomethacin, and these effects occurred in a dose‑ and time‑dependent manner. This inhibition was due to the release of second mitochondria‑derived activator of caspase (Smac) into the cytosol and the activation of caspase‑3. Subsequently, flow cytometry was performed to investigate indomethacin‑induced apoptosis following the overexpression or knockdown of Smac, and western blot analysis was performed to determine the expression of Smac and the activation of caspase‑3. Overexpression of Smac was promoted apoptosis, while downregulation of Smac significantly inhibited apoptosis. Western blot analysis demonstrated that indomethacin induced apoptosis through releasing Smac into the cytosol and activating caspase‑3. These results indicated that Smac is essential for the apoptosis induced by indomethacin in esophageal cancer cells.

Effects of endostar on growth of human esophageal carcinoma EC9706 cells and the expression of hypoxia inducible factor-1αand vascular endothelial growth factor

Objective To evaluate the effects of endostar on the growth of human esophageal carcinoma EC9706 cells and the expression of hypoxia inducible factor(HIF)-1αand vascular endothelial growth factor (VEGF). Methods EC9706 cells were treated with different cincentrations(12. 5, 25. 0, 50. 0, 100. 0 and 200. 0μg/L)of endostar for 24,48 and 72 h.The cell growth inhibition was assessed by methyl thiazol tetrazolium(MTT)assay.The expression of HIF-1αand VEGF mRNA and protein in EC9706 cells was detected by reverse transcriptase-polymerase chain reaction(RT-PCR)and immunocytochemistry respectively. Results Endostar could suppress the growth of EC9706 cells in a dose-and time-dependent response,and the inhibitive rate was 46. 78% by 200μg/L endostar for 72 h.As compared with control group, the absorbance(A)values of HIF-1αand VEGFin different groups(25. 0,50. 0,100. 0μg/L endostar)was(0. 115 ±0. 009),(0. 089±0. 011)and(0. 056±0. 009), and(0. 137±0. 007),(0. 102±0. 008)and(0. 063± 0. 007), respectlively, P< 0. 05. As compared with control group, the HIF-1αand VEGF mRNA expression levels in EC9706 cells were down-regulated after treatment with different cincentrations(25. 0, 50. 0, 100. 0μg/L) of endostar(0. 469±0. 530, 0. 233±0. 280 and 0. 171±0. 220, and 0. 304± 0. 410, 0. 199±0. 520 and 0. 071±0. 310, respectlively, P< 0. 05).Spearman correlation analysis showde a positive correlation between the HIF-1αand VEGF protein and m RNA expression. Conclusion Endostar can suppress the growth of EC9706 cells probably by further down-regulating the expression of HIF-1αand VEGF. Key words: Esophageal carcinoma; Cell proliferation; Endostar; Hypoxia inducible factor-1α; Vascular endothelial growth factor

Bardoxolone methyl induces apoptosis and autophagy and inhibits epithelial-to-mesenchymal transition and stemness in esophageal squamous cancer cells.

Natural and synthetic triterpenoids have been shown to kill cancer cells via multiple mechanisms. The therapeutic effect and underlying mechanism of the synthetic triterpenoid bardoxolone methyl (C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; CDDO-Me) on esophageal cancer are unclear. Herein, we aimed to investigate the anticancer effects and underlying mechanisms of CDDO-Me in human esophageal squamous cell carcinoma (ESCC) cells. Our study showed that CDDO-Me suppressed the proliferation and arrested cells in G2/M phase, and induced apoptosis in human ESCC Ec109 and KYSE70 cells. The G2/M arrest was accompanied with upregulated p21Waf1/Cip1 and p53 expression. CDDO-Me significantly decreased B-cell lymphoma-extra large (Bcl-xl), B-cell lymphoma 2 (Bcl-2), cleaved caspase-9, and cleaved poly ADP ribose polymerase (PARP) levels but increased the expression level of Bcl-2-associated X (Bax). Furthermore, CDDO-Me induced autophagy in both Ec109 and KYSE70 cells via suppression of the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway. There were interactions between the autophagic and apoptotic pathways in Ec109 and KYSE70 cells subject to CDDO-Me treatment. CDDO-Me also scavenged reactive oxygen species through activation of the nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) pathway in Ec109 and KYSE70 cells. CDDO-Me inhibited cell invasion, epithelial–mesenchymal transition, and stemness in Ec109 and KYSE70 cells. CDDO-Me significantly downregulated E-cadherin but upregulated Snail, Slug, and zinc finger E-box-binding homeobox 1 (TCF-8/ZEB1) in Ec109 and KYSE70 cells. CDDO-Me significantly decreased the expression of octamer-4, sex determining region Y-box 2 (Sox-2), Nanog, and B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1), all markers of cancer cell stemness, in Ec109 and KYSE70 cells. Taken together, these results indicate that CDDO-Me is a promising anticancer agent against ESCC. Further studies are warranted to explore the molecular targets, efficacy and safety of CDDO-Me in the treatment of ESCC.

Trichostatin A, a histone deacetylase inhibitor, suppresses proliferation and promotes apoptosis of esophageal squamous cell lines.

Histone deacetylase (HDAC)‑mediated epigenetic modification plays crucial roles in numerous biological processes, including cell cycle regulation, cell proliferation and apoptosis. HDAC inhibitors demonstrate antitumor effects in various cancers, including glioblastoma and breast cancer. HDAC inhibitors are therefore promising antitumor drugs for these tumors. The tumorigenesis and development of esophageal squamous cell carcinoma (ESCC) involve genetic and epigenetic mechanisms. However, the effects of the HDAC inhibitor on ESCC are not fully investigated. In the present study, ESCC cells were treated with trichostatinA (TSA) and its antitumor effects and related mechanisms were investigated. The results indicated that TSA suppressed the proliferation of ESCCs and caused G1 phase arrest by inducing the expression of p21 and p27. TSA also induced cell apoptosis by enhancing the expression of pro‑apoptotic protein Bax and decreasing the expression of anti‑apoptotic protein Bcl‑2. Furthermore, TSA inhibited the expression of phosphatidylinositol‑3‑kinase (PI3K) and reduced the phosphorylation of Akt and extracellular signal‑regulated kinase (ERK)1/2 in EC9706 and EC1 cell lines. High levels of acetylated histone H4 were detected in TSA‑treated ESCC cell lines. Overall, these results indicate that TSA suppresses ESCC cell growth by inhibiting the activation of the PI3K/Akt and ERK1/2 pathways. TSA also promotes cell apoptosis through epigenetic regulation of the expression of apoptosis‑related protein.

Peroxiredoxin 1 promotes tumorigenesis through regulating the activity of mTOR/p70S6K pathway in esophageal squamous cell carcinoma.

The biological function of Peroxiredoxin 1 (Prdx1) in cancer is still ambiguous, and its mechanism has not been elucidated so far. Previous studies have shown that Prdx1 functions as tumor suppressor in several types of cancers, but other studies have indicated that it is overexpressed in some types of human cancers, and inhibition of Prdx1 by shRNA contributes to radiosensitivity and chemosensitivity. In this study, a suppression subtractive hybridization cDNA library between esophageal squamous cell carcinoma (ESCC) cell line EC9706 and noncancerous esophageal epithelial cell line Het-1A was constructed, and 11 tumorigenesis-associated genes including Prdx1 were isolated. In addition, we further confirmed that Prdx1 was overexpressed in ESCC cells at the level of protein compared with Het-1A (P<0.05). Inhibition of Prdx1 by shRNA lentivirus decreased cell proliferation and invasion, and induced cell apoptosis, but did not affect cell cycle distribution of EC9706 cells (P>0.05). Importantly, the total proteins of mTOR and p70S6K, as well as the activity of mTOR/p70S6K signaling pathway, were decreased in Prdx1-depletion EC9706 cells. Furthermore, the activity of mTOR/p70S6K signaling pathway was increased in Prdx1-overexpressing Het-1A cells. These findings mentioned above demonstrate that Prdx1 may be involved in tumorigenesis through regulation of mTOR/p70S6K pathway in ESCC.

Silencing stathmin-modulating efficiency of chemotherapy for esophageal squamous cell cancer with paclitaxel.

Paclitaxel (PTX) is broadly considered the drug of choice for treating human esophageal squamous cell cancer (ESCC). However, PTX resistance often ultimately leads to treatment failure. stathmin, or Op18, is a ubiquitously expressed 19-kDa cytosolic phosphoprotein that can integrate various cellular regulatory signals. stathmin overexpression could lead to resistance to chemotherapeutic agents. In this study we investigated the effect of stathmin gene silencing, using small interfering RNA (stathmin siRNA), on the efficacy of PTX in ESCC. Transfection of stathmin siRNA could significantly inhibit stathmin mRNA and protein levels in ESCC cell lines EC9706 and Eca-109. The silencing of stathmin combined with PTX significantly inhibited the proliferation of EC9706 and Eca-109 cells, with a significantly higher proportion of cells at G2/M phase and this antiproliferative effect was accompanied by an increase in apoptosis rates and morphology changes of EC9706 and Eca-109. Thus, combined chemotherapeutic agent PTX and stathmin siRNA could potentially enhance the therapeutic outcomes of PTX in treating ESCC.