Esophageal Cancer Cell Line Eca-109 Research Articles

Expression of miRNA-22 in esophageal and gastric cancer tissues and cell lines

Objective To investigate the expression and clinical value of miRNA-22 in esophageal and gastric cancer tissues and cell lines. Methods Realtime-PCR was performed to detect the expression of miRNA-22 in human esophageal cancer cell lines ECA109, TE-1, TE-8, TE-13 and normal esophageal epithelial cells HEEC, 48 cases of esophageal cancer tissues and matched adjacent normal tissues, human gastric cancer cell lines SGC-7901, MKN-45, NCI-N87, AGS, NUGC-3, SUN-1, normal human gastric mucosa cell line GES-1 and normal stomach fibroblastic cell NSFC, 88 cases of gastric cancer tissues and matched adjacent normal tissues. Results The expression of miRNA-22 was much less in four esophageal cancer cell lines than that of immortalized normal esophageal epithelial cells HEEC, and the expression of miRNA-22 was much less in six gastric cancer cell lines than that of gastric mucosal epithelial cell line GES-1 and normal stomach fibroblastic cell line NSFC.The esophageal cancer tissues showed aberrant down regulation of miRNA-22 compared with the adjacent non-tumor tissues [(3.51±1.05) vs.(11.23±2.95), t=18.13, P<0.05]. The gastric cancer tissues showed aberrant down regulation of miRNA-22 compared with the adjacent non-tumor tissues [(2.15±1.23) vs.(9.14±2.86), t=22.93, P<0.05]. Conclusion The expression of miRNA-22 was much lower in esophageal and gastric cancer tissues and cell lines, which provided basement to nosogenesis, early diagnosis and create drug treatment of the cancers. Key words: Esophageal neoplasms; Gastric neoplasms; Cell lines; microRNA-22

微小RNA-143抑制食管癌细胞株ECA109的增殖、迁移和侵袭

Objective To investigate the functions of microRNA-143 (miR-143) in esophageal cancer cell line ECA109. Methods ECA109 cells were transfected with negative control (NC), miR-143 mimics or miR-143 inhibitors. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed to evaluate the growth of ECA109 cells after transfection. Annexin V-FITC/PI apoptosis test kit was used to detect early apoptosis rate in ECA109 cells. Transwell migration and invasion assays were conducted to compare the migration and invasion capacity of ECA109 among different groups. Real-time PCR and Western blotting were used to analyze the mRNA and protein alteration after transfection. Results Three and four days after transfection, compared with NC (absorbance value: 0.90±0.02 and 1.09±0.07), miR-143 mimics inhibited ECA109 cell proliferation (absorbance value: 0.66±0.05 and 0.80±0.04), while miR-143 inhibitors promoted cell proliferation (absorbance value: 1.13±0.09 and 1.51±0.08), with statistical significances (F=49.16, P=0.000; F=100.34, P=0.000). Early-stage apoptosis rates of ECA109 transfected with NC, miR-143 mimics and miR-143 inhibitors were 3.42%±0.72%, 11.63%±1.15% and 0.94%±0.10%, respectively, with statistical significance (F=151.61, P=0.000). Meanwhile, compared with NC (migration cell number: 336±13, invasion cell number: 147±16), miR-143 mimics inhibited cell migration (148±16) and invasion (75±10), while miR-143 inhibitors promoted cell migration (510±14) and invasion (238±16), with statistical significances (F=470.99, P=0.000; F=90.04, P=0.000). Compared with NC (1.00±0.00), miR-143 mimics down-regulated mRNA (relative expression level 0.22±0.08) and protein expression (relative expression level 0.46±0.08) of K-ras, whereas miR-143 inhibitors up-regulated mRNA (1.55±0.12) and protein expression (1.33±0.05) of K-ras (F=131.36, P=0.000; F=88.17, P=0.000). Conclusion miR-143 functions as a tumor suppressor in esophageal cancer cell line ECA109, probably by down-regulating K-ras expression. Key words: MicroRNAs; Esophageal neoplasms; Genes, tumor suppressor

Novel BRCA2-Interacting Protein, LIMD1, Is Essential for the Centrosome Localization of BRCA2 in Esophageal Cancer Cell.

Mutation of breast cancer 2, early onset (BRCA2) has been identified as a vital risk factor for esophageal cancer (EC). To date, several proteins have been reported as BRCA2-interacting proteins and are associated with multiple biological processes. This study’s aim was to identify a novel interactive protein of BRCA2 and to explore its functional roles in EC. A yeast two-hybrid screening was performed to identify a novel BRCA2-interacting protein. Glutathione-S-transferase (GST) pull-down analysis was performed to find out how the binding domain of BRCA2 interacts with LIM domains containing 1 (LIMD1). The interaction between LIMD1 and BRCA2 at the endogenous level was confirmed by using coimmunoprecipitation and immunobloting. Furthermore, two different sequences of short hairpin RNAs (shRNAs) against LIMD1 were transfected into the human EC cell line ECA109. Afterward, the effects of LIMD1 suppression on the centrosome localization of BRCA2 and cell division were analyzed using an immunofluorescence microscope. Results showed that LIMD1 was a novel BRCA2-interacting protein, and LIMD1 interacted with the conserved region of BRCA2 (amino acids 2,750–3,094) in vitro. Importantly, after interfering with the protein expression of LIMD1 in ECA109 cells, the centrosome localization of BRCA2 was significantly abolished and abnormal cell division was significantly increased. These results suggested that LIMD1 is a novel BRCA2-interacting protein and is involved in the centrosome localization of BRCA2 and suppression of LIMD1, causing abnormal cell division in EC cells.

Encoding gene mutation of mitochondrial DNA in esophageal cancer cells

Objective To investigate the relationship of mitochondrial DNA (mtDNA) encoding gene mutation with the occurrence and development of esophageal cancer. Methods MtDNA was extracted in three kinds of human esophageal cancer cell lines EC9706, TE-1 and Eca109, and normal esophagus cell line, Het-1A. All of the encoding genes were sequenced. Results There were 39 mutations in three kinds of esophageal cancer cells, and 11 mutations in Het-1A cells. Most of the mutations were A-G or C-T. A total of 11 protein coding mutations have been identified. Except A8860G and A15326G, the two mutations appeared in normal esophageal cells at the same time, and the remaining 9 mutations only appeared in cancer cells (G6366A, A8701G, A10086G, A10398G, A12358G, A13105G, C14766T, A15311G, and A15824G). The corresponding mutation genes were cytochrome c oxidase subunit 1 (COX1), ATP synthase 6 (ATP6), nicotinamide adenine dinucleotide dehydrogenase 3 (ND3), ND3, nicotinamide adenine dinucleotide dehydrogenase 5 (ND5), ND5, cytochromes B (CytB), CytB, CytB. Conclusion The mutations of the mtDNA encoding gene of esophageal cancer cells were related to the occurrence and development of esophageal cancer. Key words: Esophageal cancer; Mitochondrial DNA; Encoding gene; Mutation

Effects of siRNA-Mediated Knockdown of HDAC1 on the Biological Behavior of Esophageal Carcinoma Cell Lines.

BackgroundHDAC1 has been shown to be closely associated with the occurrence of tumors. We aimed to investigate the effects of siRNA-mediated HDAC1 knockdown on the biological behavior of esophageal carcinoma cell lines.Material/MethodsHDAC1 expression in esophageal cancer cell lines TE-1, Eca109, and EC9706 was compared by Western blot analysis. These cells were transfected with siRNA-HDAC1 and cell proliferation was evaluated by MTT assay to select the optimum cell line for subsequent experiments. The effects of siRNA-HDAC1 on the migration and invasion of the selected cell line were assessed by transwell assay. The expression of cell cycle-related proteins cyclinD1, p21 and p27, and epithelial-mesenchymal transition (EMT)-related protein zonula occludens-1 (ZO-1), E-cadherin and vimentin was determined by Western blot analysis.ResultsHDAC1 expression in TE-1, Eca109 and EC9706 cells was significantly higher compared with normal esophageal cell line HEEC (P<0.01). MTT assay, Western blot and RT-PCR analyses demonstrated that the inhibitory effects of siRNA on HDAC1 expression and cell viability in TE-1 cells were the highest among all cell lines, which was therefore used in subsequent experiments. After TE-1 cells were transfected with siRNA-HDAC1, their migration and invasion were significantly lower compared with the controls (P<0.01). CyclinD1 and vimentin expression was significantly lower compared with the controls (P<0.01), whereas the expression of p21, p27, ZO-1 and E-cadherin was significantly higher (P<0.01).ConclusionsThe siRNA-mediated HDAC1 knockdown significantly inhibited the proliferation, migration and invasion of TE-1 cells probably by regulating the expression of cell cycle- and EMT-related proteins.

Expression and significance of FoxM1 in esophageal squamous cell carcinoma cells in vitro and in vivo

To investigate the expression and significance of FoxM1 in esophageal squamous cell carcinoma (ESCC) cell lines and tissues. Western blot assay was used to detect the expression of FoxM1 in human esophageal epithelial cells and esophageal squamous cell cancer cell lines TE1, TE10, TE11 and Eca109 cells. To determine whether down-regulation of FoxM1 expression could inhibit the aggressive phenotype of ESCC cells, we knocked down the expression of FoxM1 by using FoxM1-shRNA in TE1 cells. Then we detected the cell proliferation, migration and invasion of TE1 cells by MTT assay, scratch assay and transwell assay. Furthermore, the effect of FoxM1 knockdown on tumorigenicity in nude mice was evaluated. Finally, immunohistochemical staining was used to detect the expression of FoxM1 in 99 cases of ESCC tissues and adjacent normal esophageal tissues. χ(2) test was used to analyze the correlations between the expression of FoxM1 and clinicopathologic characteristics and prognosis of ESCC patients. Western blot data showed that FoxM1 expression was lower in normal esophageal epithelial cells and highly expressed in four esophageal cancer cell lines, especially in TE1 cells. Knockdown of FoxM1 inhibited the growth, invasion and migration of TE1 cells and reduced their tumorigenicity in nude mice.The positive expression rate of FoxM1 in ESCC was 61.6% (61/99), significantly higher than that in the paired adjacent normal tissues (24.2%, 24/99) (P<0.05). The positive expression rate of FoxM1 in ESCC tissues was 61.6% (61/99), significantly higher than that in the paired adjacent normal tissues (24.2%, 24/99) (P<0.05). FoxM1 expression was significantly and positively correlated with lymph node metastasis, clinical stage and invasive depth (P<0.05). The median survival time was 42.3 months in 38 cases of patients with negative FoxM1 expression, and 33.0 months in 61 cases of positive FoxM1 expression, and the difference was statistically significant (P=0.036). FoxM1 is highly expressed in ESCC, and significantly correlated with the initiation, development and prognosis of esophageal cancer. FOXM1 might be an indicator to predict the prognosis and serve as a potential target for therapy in esophageal cancer.

FGF2 regulates proliferation, migration, and invasion of ECA109 cells through PI3K/Akt signalling pathway in vitro.

Esophageal cancer is one of the most common malignant cancers that arise from esophagus tissues. Fibroblast growth factor 2 (FGF2) has been implicated in multiple biological functions and was considered as an oncogenic factor in tumorigenesis. However, the effects of FGF2 in esophageal carcinoma are yet to be fully elucidated. To better understand the function of FGF2 in esophageal cancer, we used the esophageal cancer cell line ECA109 as a cell model and downregulated FGF2 expression using RNAi; the results showed that insufficient expression of FGF2 inhibited cells proliferation, migration, and invasion of ECA109 cells. Meanwhile, the proliferation, migration, and invasion abilities were stimulated after treatment of exogenous FGF2. In addition, a PI3K/Akt signalling pathway inhibitor (LY294002) alleviated the tumorigenic effects of FGF2. These findings implied that the oncogenic effects of FGF2 was mediated, at least in part, through the PI3K/Akt signalling pathway and FGF2 may be a potential therapeutic target to constrain the tumorigenesis of esophageal cancer.

Antitumor effects evaluation of a novel porphyrin derivative in photodynamic therapy.

In this paper, the antitumor activity of a novel porphyrin-based photosensitizer 5,10,15,20-tetrakis[(5-diethylamino)pentyl] porphyrin (TDPP) was reported in vitro and in vivo. The photophysical and cellular properties of TDPP were investigated. The singlet oxygen generation quantum yield of TDPP was detected; it showed a high singlet oxygen quantum yield of 0.52. The intracellular distribution of photosensitizer was detected with laser scanning confocal microscopy. The efficiency of TDPP-photodynamic therapy (PDT) in vitro was analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and in situ trypan blue exclusion test. Treated with a 630-nm laser, TDPP can kill cultured human esophageal cancer cell line (Eca-109) cells and reduce the growth of Eca-109 xenograft tumors significantly in BABL/c nude mice. And histopathological study was also used to confirm the antitumor effect. It has the perspective to be developed as a new antitumor drug in photodynamic therapy and deserves further investigation.

HIF-1α induces VE-cadherin expression and modulates vasculogenic mimicry in esophageal carcinoma cells.

To investigate whether hypoxia inducible factor (HIF)-1α modulates vasculogenic mimicry (VM) by upregulating VE-cadherin expression in esophageal squamous cell carcinoma (ESCC). Esophageal squamous cancer cell lines Eca109 and TE13 were transfected with plasmids harboring small interfering RNAs targeting HIF-1α or VE-cadherin. The proliferation and invasion of esophageal carcinoma cells were detected by MTT and Transwell migration assays. The formation of tubular networks of cells was analyzed by 3D culture in vitro. BALB/c nude mice were used to observe xenograft tumor formation. The relationship between the expression of HIF-1α and VE-cadherin, ephrinA2 (EphA2) and laminin5γ2 (LN5γ2) was measured by Western blot and real-time polymerase chain reaction. Knockdown of HIF-1α inhibited cell proliferation (32.3% ± 6.1% for Eca109 cells and 38.6% ± 6.8% for TE13 cells, P < 0.05). Both Eca109 and TE13 cells formed typical tubular networks. The number of tubular networks markedly decreased when HIF-1α or VE-cadherin was knocked down. Expression of VE-cadherin, EphA2 and LN5γ2 was dramatically inhibited, but the expression of matrix metalloproteinase 2 had no obvious change in HIF-1α-silenced cells. Knockdown of VE-cadherin significantly decreased expression of both EphA2 and LN5γ2 (P < 0.05), while HIF-1α expression was unchanged. The time for xenograft tumor formation was 6 ± 1.2 d for Eca109 cells and Eca109 cells transfected with HIF-1α Neo control short hairpin RNA (shRNA) vector, and 8.4 ± 2.1 d for Eca109 cells transfected with an shRNA against HIF-1α. Knockdown of HIF-1α inhibited vasculogenic mimicry (VM) and tumorigenicity in vivo. HIF-1α may modulate VM in ESCC by regulating VE-cadherin expression, which affects VM formation through EphA2 and LN5γ2.

Andrographolide radiosensitizes human esophageal cancer cell line ECA109 to radiation in vitro.

To explore the radiosensitivity of andrographolide on esophageal cancer cell line ECA109. The inhibition effects of andrographolide were measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Clonogenic survival assay was used to evaluate the effects of andrographolide on the radiosensitivity of esophageal cancer cells. Immunofluorescence was employed to examine Bax expression. The changes in cell cycle distribution and apoptosis were assayed using flow cytometry. The expression of NF-κb/Cleaved-Caspase3/Bax/Bcl-2 was measured using Western blot analysis. DNA damage was detected via γ-H2AX foci counting. With a clear dose and time effects, andrographolide was found to inhibit the proliferation of esophageal cell line ECA109. The results of the clonogenic survival assay show that andrographolide could markedly enhance radiosensitivity (P < 0.05) with a sensitizing enhancement ratio of 1.28. Andrographolide caused a dose-dependent increase in Cleaved-Caspase3/Bax protein expression and a decrease in Bcl-2/NF-κb expression. Apoptosis in andrographolide-treated ECA-109 increased significantly compared with the apoptosis in the simple drug and radiation combined with drug groups (P < 0.001; P < 0.05). Moreover, compared with the independent radiation group, the andrographolide combined with radiation group increased the number of DNA double chain breaks. Andrographolide can increase the radiosensitivity of esophageal cell line ECA109. This result may be associated with the decrease in the NF-κb level and the induced apoptosis of esophageal cancer cells.

BTG1 underexpression is an independent prognostic marker in esophageal squamous cell carcinoma.

To determine the expression and function of B cell translocation gene 1 (BTG1) in esophageal carcinoma, esophageal samples were taken from cancer lesions (n = 74) and adjacent normal tissue (n = 34) in esophageal cancer patients immediately after endoscopic biopsy. BTG1 expression was determined by immunohistochemistry and Western blotting. The effect of BTG1 overexpression was examined in vitro utilizing a human esophageal cancer cell line ECA-109 stably transfected with a recombinant lentivirus (LeBTG1 cells) and compared to empty vector-transfected controls (LeEmpty). BTG1 overexpression was verified by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot. The expression of proteins involved in cell cycle regulation (cyclin D1) and apoptosis (Bcl-2) and cell migration (MMP-9) in LeBTG1 cells was analyzed by Western blot. The effect of BTG1 overexpression on cell viability and proliferation was assessed by an MTT assay in LeBTG1 and LeEmpty cells. Flow cytometric analyses were used to evaluate the effect of BTG1 expression on cell cycle distribution and apoptosis. The migration and invasion potential of LeBTG1 cells was examined by plating cells in Matrigel-coated chambers. The level of BTG1 protein expression was found to be significantly lower in esophageal cancer tissue than normal tissues (P < 0.05). Decreased expression of BTG1 was significantly correlated with lymph node metastasis, clinical stage, and histological grade of patients with esophageal cancer (P < 0.05). Meanwhile, loss of BTG1 expression correlated significantly with poor overall survival time by Kaplan-Meier analysis (P < 0.05). The result of biological function shown that Eca-109 cell-transfected BTG1 had a lower survival fraction, higher percentage of the G0/G1 phases, higher cell apoptosis, significant decrease in migration and invasion, and lower cylin D1, Bcl-2, and MMP-9 protein expression compared with Eca-109 cell-untransfected BTG1 (P < 0.05). Reduced BTG1 expression is associated with increased disease severity, suggesting it is a negative regulator of esophageal cancer and can serve as a prognostic indicator.

Human mesenchymal stem cells with adenovirus-mediated TRAIL gene transduction have antitumor effects on esophageal cancer cell line Eca-109.

The apoptotic ligand TNF-related apoptosis-inducing ligand (TRAIL) is believed to be a promising candidate for cancer gene therapy, yet gene therapy strategies to tackle this disease systemically are often impaired by inefficient delivery of the vector to the tumor tissue. Mesenchymal stem cells (MSCs) have been shown to home to tumor sites and could potentially act as a shield and vehicle for an antitumor gene therapy vector. Here, we used an adenoviral vector expressing TRAIL to transduce MSCs and studied the apoptosis-inducing activity of these TRAIL-carrying MSCs on esophageal cancer cell Eca-109. Our results showed that, in vitro, TRAIL-expressing MSCs were able to inhibit proliferation and induce apoptosis in Eca-109 cells by an MTT assay, co-culture experiments and flow cytometry analysis. In vivo, TRAIL-expressing MSCs also displayed an ability to inhibit tumor growth in an Eca-109 xenograft mouse model. Together, our findings indicated that the gene therapy strategy of MSCs-based TRAIL gene delivery has a wide potential value for improving the treatment of esophageal cancer.

Suppression of tumor necrosis factor receptor-associated protein 1 expression induces inhibition of cell proliferation and tumor growth in human esophageal cancer cells.

Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a molecular chaperone involved in multidrug resistance and antiapoptosis in some human tumors, but its regulatory mechanisms have not been revealed in esophageal squamous cell carcinoma (ESCC). In this study, 138 specimens of ESCC were analyzed. TRAP1 was overexpressed in ESCC, particularly in poorly differentiated tumors. To further explore the molecular regulatory mechanism, we constructed specific small interfering RNA-expressing vectors targeting Trap1, and knocked down Trap1 expression in the esophageal cancer cell lines ECA109 and EC9706. Knockdown of Trap1 induced increases in reactive oxygen species and mitochondrial depolarization, which have been proposed as critical regulators of apoptosis. The cell cycle was arrested in G2/M phase, and in vitro inhibition of cell proliferation was confirmed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and bromodeoxyuridine assays. Furthermore, re-expression of TRAP1 in Trap1 small interfering RNA-transfected ESCC cells restored cell proliferation and cell apoptosis. Bioluminescence of subcutaneously xenografted ESCC tumor cells demonstrated significant inhibition of in vivo tumor growth by Trap1 knockdown. This study shows that TRAP1 was overexpressed in most patients with ESCC, and caused an increase in antiapoptosis potency. TRAP1 may be regarded as a target in ESCC biotherapy.

Dihydroartemisinin Accentuates the Anti-Tumor Effects of Photodynamic Therapy via Inactivation of NF-κB in Eca109 and Ec9706 Esophageal Cancer Cells

Background: Photodynamic therapy (PDT) is a new treatment for esophageal cancer which has been shown to be effective in the elimination of tumor. However, PDT could induce the activation of nuclear factor-kappa B (NF-κB) in many photosensitizers based PDT, which plays a negative role in PDT. In addition, our previous results have shown that dihydroartemisinin (DHA), which was the most potent one of artemisinin derivatives, has anticancer activity in esophageal cancer cells. Methods: Cell viability was determined by MTT analysis, and apoptosis was evaluated by flow cytometry. Nuclear extract was obtained for determining NF-κB DNA-binding activity, while total protein extract obtained for downstream gene expression by western blot. Results: We demonstrated DHA enhanced PDT-induced growth inhibition and apoptosis in both human esophageal cancer cell lines Eca109 and Ec9706 in vitro. The mechanism was at least partially due to DHA deactivated PDT-induced NF-κB activation, so as to decrease tremendously the expression of its target gene Bcl-2. Conclusion: Our results demonstrate that DHA augments PDT-induced growth inhibition and apoptosis in esophageal cancer cells, and that inactivation of NF-κB activity is a potential mechanism by which DHA sensitizes esophageal cancer cells to PDT-induced growth inhibition and apoptosis.

P162 increases radiosensitivity of esophageal cancer cell line Eca109 by inhibiting Chk1/2 expression

P162 increases radiosensitivity of esophageal cancer cell line Eca109 by inhibiting Chk1/2 expression

The radiation-sensitizing effect of flavopiridol in the esophageal cancer cell line Eca109

Flavopiridol is a cyclin-dependent kinase inhibitor. It has shown an antitumor effect against several cancers. In the present study, the radiation-sensitizing effect of flavopiridol was investigated in an esophageal squamous carcinoma cell line, Eca109. The growth inhibitory rate of Eca109 with flavopiridol was determined using the MTT and the radio-sensitizing rate using clonogenic survival assays. The cell cycle distribution and the rate of apoptosis were measured using flow cytometry. The proteins cyclin D1, ERK/pERK, caspase-3, Bax and Bcl-2 were detected using western blot analysis to elucidate the mechanism of the radiosensitization effect. MTT assay showed that flavopiridol inhibited the survival rate of Eca109 cells and the effect was dose-dependent. Its IC50 was 193.3 nmol/l. The result of the clonogenic survival revealed that flavopiridol enhanced the radiosensitivity of Eca109 cells and the sensitization enhancement ratio (SER) was 1.194 at 0.2×IC50. Moreover, we detected that the cells treated with flavorpiridol were arrested at the G2/M phase and the apoptosis caused by radiation was increased. In addition, the proteins caspase-3 and Bax in cells treated with flavopiridol were upregulated, while cyclin D1 and Bcl-2 were downregulated. In conclusion, flavopiridol may enhance the radiosensitivity of Eca109 cells and the radiosensitizing effect of flavopiridol may be mediated by decreasing the levels of the cyclin D1 protein, thus increasing the percentage of cells at G2/M phase.

Roles of phosphorylated JNK in esophageal squamous cell carcinomas of kazakh ethnic

The c-Jun NH2 -terminal kinase (JNK) signal pathway has been implicated in the growth, cellular proliferation, and apoptosis in many kinds of carcinomas. However, the role of JNK in the development of esophageal squamous cell carcinomas (ESCCs) is unknown. To investigate the role of JNK in ESCC, in vitro, esophageal cancer cell line Eca109 was pretreated using SP600125, JNK specific inhibitor, then was subjected to MTT assay to examine cellular proliferation, flow cytometric analysis to detect apoptosis and cell cycle, and wound healing assay to evaluate cell migration. Meanwhile, the mRNA and protein expression of JNK in Eca109 cells pretreated with SP600125 were examined by real-time quantitative reverse transcription PCR (qRT-PCR) and Western blotting, respectively. In vivo, 12 paired of fresh ESCC and normal adjacent tissues (NAT) from Kazakh patients were used to validate the expression of JNK by qRT-PCR and Western blotting. Furthermore, to reconfirm the expression trend of activation JNK (p-JNK), enlarged 72 paired of Kazakh's ESCC and NAT were subjected to immunohistochemistry. Our results showed that the suppression of p-JNK could lead to apoptosis and reduce proliferation in Eca109 cells. However, there was an elevated expression of p-JNK protein in NAT compared with ESCC tissues, and there was significant difference between p-JNK expression and pathological differentiation (P < 0.05) in Kazakh populations. Together, all the data we obtained in the present study indicated that the p-JNK MAPK pathway was involved in pathogenesis of Kazakh's ESCC, and played a different roles in carcinogenesis and development of Kazakh's ESCC.

LMP gene promoter hypermethylation is a mechanism for its down regulation in Kazak’s esophageal squamous cell carcinomas

Abnormal hypermethylation of CpG islands not only associated with tumor suppressor genes can lead to repression of gene expression, but also contribute to escape of the tumor from immune surveillance and contribute significantly to tumorigenesis. In the present study, we studied the hypermethylation of low molecular-weight protein (LMP) gene and its regulation on protein expression in biopsies from resected tissues from Kazak's esophageal squamous cell carcinoma (ESCC) patients and their neighboring normal tissues. LMP2 and LMP7 genes promoter region methylation sequences were maped in esophageal cancer cell line Eca109 by bisulfite-sequencing PCR and quantitative detection of methylated DNA from 30 pairs of Kazak's ESCC and adjacent normal tissues by MassARRAY (Sequenom, San Diego, CA, USA) and LMP2 and LMP7 protein expression were analyzed with immunohistochemistry. In Eca109, we identified 6 CG sites methylated from all of 22 CpG sites of LMP7 gene. However, no methylation was found for LMP2. The analysis of the data resulted from the quantitative analysis of single CpG site methylation by Sequenom MassARRAY platform, has shown that the methylation level between two groups CpG sites (CpG_5, CpG_9, CpG_20, CpG_21 and CpG_20) from CpG_1, CpG_2, CpG_3, CpG_4, CpG_5, CpG_6, CpG_7, CpG_8, CpG_9, CpG_10.11, CpG_12.13.14, CpG_15.16.17.18, CpG_19, CpG_20, CpG_21 and CpG_22 significant differences between ESCC and neighboring normal tissues. The analysis of methylation level of whole target CpG fragment indicated that the methylation level of LMP7 was significant higher in ESCC (0.0517 ± 0.0357) than in neighboring normal tissues (0.0380 ± 0.0214, P < 0.05). there was a tendency of decreasing the LMP7 proteins expression as the increasing the methylation level of LMP7 gene promoter regions (F = 7.69, P = 0.041). The LMP7 gene promoter methylation and protein downregulation were correlated at high extent in Kazakh's ESCC patients, and may explain the epigenetic regulation on gene expression.

Effects of metformin on cell proliferation and apoptosis in human esophageal squamous cancer cell line Eca109

Effects of metformin on cell proliferation and apoptosis in human esophageal squamous cancer cell line Eca109

OCT4 Positively Regulates Survivin Expression to Promote Cancer Cell Proliferation and Leads to Poor Prognosis in Esophageal Squamous Cell Carcinoma

BackgroundOCT4 and Survivin are important factors for cancer cell proliferation, renewal and dedifferentiation, and correlate with resistance to radiotherapy and chemotherapy in most human cancers, but their regulatory mechanisms are not well known.Methodology/Principal FindingsIn this study, 50 patients with esophageal squamous cell carcinoma (ESCC) were retrospectively analyzed. OCT4 was expressed in 13 cases (26%), and survivin was positively expressed in 31 cases (62%), examined by immunochemistry. OCT4 was found to be an independent predictive factor for median survival time, and the patients from the subgroup with both high expression of OCT4 and Survivin had the worst prognosis investigated by log-rank test. To further explore the molecular regulatory mechanism between OCT4 and Survivin, we constructed the specific small hairpin RNA (shRNA)-expressing vectors targeting OCT4 or/and Survivin and manipulated the expression of OCT4 and Survivin. By Western blotting and RT-PCR, we found that OCT4 could up-regulate Survivin expression in the esophageal cancer cell lines Eca109 and TE1. Simultaneously knockdown of OCT4 and Survivin expression induced cell apoptosis and G2-phase decrease of cell cycle by flow cytometry, and finally exerted an enhanced anti-proliferation potency in Eca109 and TE1 cell lines by MTT assay.ConclusionsThis study shows that OCT4 and Survivin expression were correlated with poor survival in patients with ESCC. OCT4 and Survivin may be regarded as targets in ESCC biotherapy.