Esophageal Cancer Cell Line EC9706 Research Articles

Relationship Between PTEN and Angiogenesis of Esophageal Squamous Cell Carcinoma and the Underlying Mechanism.

Esophageal squamous cell carcinoma (ESCC) has high morbidity and mortality rates owing to its ability to infiltrate and metastasize. Microvessels formed in early-stage ESCC promote metastasis. Phosphatase and tensin homolog (PTEN) mediates macrophage polarization, but its effect and mechanism on early ESCC angiogenesis are unclear. To explore the molecular mechanism underlying early ESCC metastasis through blood vessels, we investigated the relationship between PTEN/phosphoinositide 3-kinase (PI3K)/p-AKT protein levels, number of infiltrated macrophages, and angiogenesis in ESCC and ESCC-adjacent normal esophageal mucosa tissues from 49 patients. Additionally, PTEN was overexpressed or silenced in the esophageal cancer cell line EC9706, and its supernatant served as conditioning medium for M1 tumor-associated macrophages (TAMs). The culture medium of macrophages served as conditioning medium for esophageal tumor-associated vascular endothelial cells (TECs) to study the biological behavior of PTEN-plasmid, PTEN-siRNA, and control TECs. We found that M1 TAM infiltration in ESCC tissues was low, whereas M2 TAM infiltration was high. Microvessel density was large, PTEN was down-regulated, and the PI3K/AKT pathway was activated in ESCC specimens. These parameters significantly related to the depth of tumor invasion, lymph node metastasis, and pathological staging of ESCC. Silencing of PTEN in EC9706 cells significantly activated the PI3K/AKT signaling pathway in macrophages, promoting M1-to-M2 TAM polarization and enhancing TECs’ ability to proliferate, migrate, invade, form tubes, and secrete vascular endothelial growth factor. We believe that PTEN silencing in esophageal cancer cells activates the PI3K/AKT signaling pathway in macrophages via the tumor microenvironment, induces M2 TAM polarization, and enhances the malignant behavior of TECs, thereby promoting ESCC angiogenesis. Our findings lay an empirical foundation for the development of novel diagnostic and therapeutic strategies for ESCC.

MiR-486 functions as a tumor suppressor in esophageal cancer by targeting CDK4/BCAS2.

Esophageal cancer is a common tumor for which morbidity and mortality are high worldwide. We aimed to study alterations in miR-486 expression in esophageal cancers, and the effect miR-486 on esophageal cancer cell function and behavior. We collected esophageal cancer tissues/corresponding normal tissues from 20 patients and utilized three esophageal cancer cell lines and normal esophageal epithelial cells, and the expression of miR-486, CDK4 and BCAS2 was detected by qRT-PCR. Western blotting was used to detect the expression of CDK4 and BCAS2 protein. Then, we overexpressed miR-486 in esophageal cancer cell line EC9706. A series of cell functional analyses, including cell growth, cell cycle, apoptosis, migration and invasion were performed in esophageal cancer cells using colony formation assay, flow cytometry, Transwell and scratch assays, respectively. Dual-Luciferase reporter gene assay was used to detect the target genes of miR-486. We found that the expression of miR-486 in esophageal cancer tissues and cell lines was significantly lower than that in the normal tissues and normal esophageal epithelial cell line. Overexpression of miR-486 significantly inhibited the colony formation ability, induced G0/G1 phase arrest and apoptosis and suppressed cell migration and invasion in the EC9706 cells. Using bioinformatics and luciferase reporter assay, we identified that CDK4 and BCAS2 may be target genes of miR-486 and levels of CDK4 and BCAS2 were both significantly higher in the esophageal cancer tissues and cell lines than levels in the normal tissues and cells. Furthermore, knockdown of CDK4/BCAS2 coincided with the suppressive effects of miR-486 in esophageal cancer cells. Expression of apoptotic signaling molecules p21 and caspase-3 was upregulated in the CDK4/BCAS2-knockdown groups. These results suggest that miR-486 may suppress tumor cell growth and metastasis in esophageal cancer by targeting CDK4/BCAS2. The newly identified miR-486/CDK4/BCAS2 pathway provides further insight into the development and progression of esophageal cancer, which is of great significance to the early diagnosis and detection of esophageal cancer.

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

Quiescence and attenuated DNA damage response promote survival of esophageal cancer stem cells

Accumulating evidence indicates cancer stem cells (CSCs) possess the capability to resist DNA-damage induced cell death, whereas the mechanism is largely unknown. Here we show that cell cycle status and DNA damage response (DDR) in CSCs probably contribute to their survival in genotoxic insults. In this study, we isolated esophageal cancer stem cells (ECSCs) from esophageal cancer cell line EC9706 by side-population (SP) phenotype through flow cytometry and found that ECSCs preferentially stay quiescent as compared to the non-ECSCs and are more resistant to DNA damage agents. Further study revealed that ECSCs express a lower level of EGFR, phosphoralated Stat3, and c-Myc, yet abnormally upregulated p27. More interestingly, different from non-ECSCs, when suffering DNA damage agents, ECSCs showed attenuated DDR, as well as declined DNA repair potential. These data indicated ECSCs probably employed an impaired DDR to handle severe genomic insults. Conclusively, we infer that the damage-resistance ability of ECSCs is likely attributed to their slow-cycling status and avoidance of apoptosis or senescence triggered by an excessive DDR.

Effect of CEA gene regulation on the anti-tumor activity of oncolytic adenovirus H101 to esophageal carcinoma

To study the effect of CEA gene regulation on the anti-tumor activity of oncolytic adenovirus H101 to esophageal carcinoma, and to explore the intrinsic factors influencing H101 sensitivity. Stable human esophageal cancer cell line EC9706 cells with lower (EC9706-SCEA) and higher CEA expression (EC9706-CEA) were chosen, thawed and cultured, and then to analyse the influence of CEA expressed at different levels on cell growth. The cytotoxic effect of H101 was assayed by in vitro and nude mouse in vivo. The cell growth experiment showed that the population doubling time of EC9706-SCEA, EC9706-CEA and EC9706 cells were (30.9 ± 2.0) h, (31.1 ± 2.5) h and (29.1 ± 2.6) h, respectively, showing no significant difference among them (P > 0.05). The cytotoxic activity of H101 was higher on EC9706-SCEA than on other four groups, when MOI was ≥ 0.01 PFU (P < 0.05). The mouse experiment showed that H101 inhibited the growth of transplanted tumors in all experimental groups. Its effect on CEA-silenced tumors (inhibition rate was 61.5% to 74.5%) was significantly higher than that on CEA-overexpression tumors (32.3% to 38.5%) and control EC9706 transplanted tumors (35.5% to 44.8%). There was a significant difference between them (P < 0.05). The results in vitro and in vivo experiments show that H101 can enhance the cytotoxic effect on EC9706 cells with lower CEA expression. To silence the expression of CEA may provide a novel strategy for target gene therapy of esophageal carcinoma.

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.

Expression of ERK1/2 MAPK signaling transduction pathway in esophageal cancers in Kazakh patients

To investigate the expression variation and significance of ERK1/2 MAPK signaling transduction pathway in the pathogenesis of esophageal squamous cell carcinoma (ESCC) in Kazakh patients. The expression level of p-ERK1/2 after serum starvation and treatment with U0126 inhibitor was detected in esophageal cancer cell line EC9706 by Western blot assay. The mRNA level of total ERK1/2 (t-ERK1/2) and expression level of t-ERK1/2 and p-ERK1/2 proteins of 25 pairs of ESCC and adjacent normal esophageal mucosal tissues of Kazakh patients were examined and identified by real-time quantitative PCR (qRT-PCR) and Western blotting, respectively. The expression of p-ERK1/2 protein was verified by immunohistochemistry in 126 paraffin-embeded specimens, including 19 normal esophageal mucosa, 55 esophageal carcinomas in situ and 52 invasive carcinomas. ERK1/2 MAPK signaling transduction pathway was in an active status in the EC9706 cells. The expression level of p-ERK1/2 in Ec9706 cells reached a peak at 10 min after transient serum stimulation, and p-ERK1/2 expression was totally restrained after the treatment with 50 µmol/L U0126. In the 25 pairs of ESCC and adjacent normal mucosa, the t-ERK1 mRNA level was 1.92 ± 3.49 in the ESCC tissues and 3.67 ± 7.47 in the adjacent normal mucosa. The t-ERK1 mRNA level in ESCC tissues was significantly lower than that in adjacent normal mucosa (P < 0.05), whereas there was no significant difference of t-ERK2 mRNA level between them(P > 0.05). The expression levels of p-ERK1 and p-ERK2 proteins were 0.87 ± 0.14 and 0.79 ± 0.10 in the ESCC tissues, and 1.10 ± 0.13 and 1.32 ± 0.12 in the adjacent normal mucosae. p-ERK1/2 protein in the ESCC tissues was significantly lower than that in the adjacent normal tissue (P < 0.01). However, there was no significant difference between their t-ERK1/2 protein levels (P > 0.05). In the 126 cases of paraffin-embeded specimens, positive expressions of both p-ERK1 and p-ERK2 in esophageal cancer tissues were 7.7% (4/52), significantly lower than those in adjacent normal mucosa (31.6%, 6/19) and carcinoma in situ (85.5%, 47/55, P < 0.05). ERK1/2 MAPK signaling pathway is in an active status in esophageal cancer and adjacent normal mucosa. Our results imply that the activation of p-ERK1/2 MAPK signaling transduction pathway plays a role in the early pathogenesis of ESCC in Kazakh patients.

The expression and activation of ERK/MAPK pathway in human esophageal cancer cell line EC9706

While there have been more and more studies concerning mitogen-activated protein kinases (MAPKs) signaling pathways, which control many cellular complex programmes, such as cell proliferation, differentiation, cell death and embryogenesis. However, few studies are carried out about expression and activation of classical MAPKs, extracellular signal-regulated kinase1/2 (ERK1/2) in human esophageal cancer cell line. Therefore, in the present study, we investigated the expression and activation of ERK1/2 in human esophageal cancer cell line EC9706 and human normal esophageal epithelial cell line Heepic, which is as control. This study showed that ERK1/2 was transiently phosphorylated both in EC9706 and Heepic, the kinetics of which were slightly different. To further study the ERK/MAPK signaling pathway in EC9706 and Heepic cell line, U0126 a kind of specific inhibitor of MEK was used. This study showed that U0126 can block the phosphorylation of ERK1/2 in a short time, the complete inhibition concentration for EC9706 and Heepic cell line is 50 and 20 μM, respectively. Incidentally, to further investigate the different roles of ERK1 and ERK2, vector-based short hairpin interference vectors targeted on ERK1/2 was constructed. Moreover, the effective interference target sequence was screened out in a transient transfection manner. MTT experiment showed that ERK2 is more important than ERK1 in the proliferation of EC9706 cells.

Resveratrol induces apoptosis in human esophageal carcinoma cells.

To investigate the apoptosis in esophageal cancer cells induced by resveratrol, and the relation between this apoptosis and expression of Bcl-2 and Bax. In in vitro experiments, MTT assay was used to determine the cell growth inhibitory rate. Transmission electron microscope and TUNEL staining method were used to quantitatively and qualitively detect the apoptosis status of esophageal cancer cell line EC-9706 before and after the resveratrol treatment. Immunohistochemical staining was used to detect the expression of apoptosis-regulated gene Bcl-2 and Bax. Resveratrol inhibited the growth of esophageal cancer cell line EC-9706 in a dose-and time-dependent manner. Resveratrol induced EC-9706 cells to undergo apoptosis with typically apoptotic characteristics, including morphological changes of chromatin condensation, chromatin crescent formation, nucleus fragmentation and apoptotic body formation. TUNEL assay showed that after the treatment of EC-9706 cells with resveratrol (10 mmol/L) for 24 to 96 hours, the AIs were apparently increased with treated time (P<0.05). Immunohistochemical staining showed that after the treatment of EC-9706 cells with resveratrol (10 mmol/L) for 24 to 96 hours, the PRs of Bcl-2 proteins were apparently reduced with treated time (P<0.05) and the PRs of Bax proteins were apparently increased with treated time (P<0.05). Resveratrol is able to induce the apoptosis in esophageal cancer. This apoptosis may be mediated by down-regulating the apoptosis-regulated gene Bcl-2 and up-regulating the expression of apoptosis-regulated gene bax.