Migration Of Eca109 Cells Research Articles

SIRT3 Promotes the Development of Esophageal Squamous Cell Carcinoma by Regulating Hexokinase 2 through the AKT Signaling Pathway.

In the present study, we explored whether sirtuin-3 (SIRT3) regulates the proliferation and migration of esophageal squamous cell carcinoma (ESCC) and investigated the mechanisms underlying the oncogene role of SIRT3. siRNA was used to transfect Eca109 cells and downregulate SIRT3. The proliferation and migration of Eca109 cells were examined by the CCK-8 assay, colony formation assay, Transwell assay, and scratch test. Quantitative real-time PCR and Western blotting were used to detect SIRT3, hexokinase 2, AKT, and p-AKT in Eca109 cells. Functional assays showed that downregulation of SIRT3 could inhibit the proliferation and migration of ESCC cells. Reduced SIRT3 expression downregulated hexokinase 2 expression and inhibited AKT activation in ESCC. These results indicated that SIRT3 promote ESCC development and progression by regulating hexokinase 2 through the AKT signaling pathway. SIRT3 promote ESCC proliferation and migration by regulating HK-2 through the AKT signaling pathway.

Essential oil from Saussurea costus inhibits proliferation and migration of Eca109 cells via mitochondrial apoptosis and STAT3 signaling

Objective: To investigate the effect and its underlying molecular mechanisms of essential oil from Saussurea costus in esophageal cancer cell line Eca109. Methods: The chemical composition of essential oil from Saussurea costus was investigated by gas chromatography-mass spectrometry (GC-MS). The anti-proliferative, anti-migrative, and apoptotic effects of essential oil from Saussurea costus against Eca109 cells were analyzed. Moreover, the expression of proteins associated with cell cycle, metastasis, and apoptosis was determined. Results: GC-MS analysis showed that essential oil from Saussurea costus was predominantly comprised of sesquiterpenes. Saussurea costus essential oil inhibited the viability of Eca109 cells in a dose-and time-dependent manner with IC50 values of (24.29±1.49), (19.16±2.27) and (6.97±0.86) μg/mL at 12, 24, and 48 h, respectively. The expression levels of target proteins in the cell cycle (phase G1/S), including cyclin D1, p21, and p53, were affected by Saussurea costus essential oil. The essential oil also downregulated the expression of metastasis-related proteins MMP-9 and MMP-2. Moreover, it induced apoptosis of Eca109 cells through the mitochondrial pathway, as well as inhibition of STAT3 phosphorylation. Conclusions: The essential oil from Saussurea costus exhibited anti-proliferative, anti-migrative, and apoptotic effects on Eca109 cells, and could be further explored as a potential anti-esophageal cancer agent.

Human Telomerase Reverse Transcriptase as a Therapeutic Target of Dihydroartemisinin for Esophageal Squamous Cancer

Objective: To elucidate the oncogenic role of human telomerase reverse transcriptase (hTERT) in esophageal squamous cancer and unravel the therapeutic role and molecular mechanism of dihydroartemisinin (DHA) by targeting hTERT. Methods: The expression of hTERT in esophageal squamous cancer and the patients prognosis were analyzed by bioinformatic analysis from TCGA database, and further validated with esophageal squamous cancer tissues in our cohort. The Cell Counting Kit-8 (CCK8) and colony formation assay were used to evaluate the proliferation of esophageal squamous cancer cell lines (Eca109, KYSE150, and TE1) after hTERT overexpression or treated with indicated concentrations of DHA. Transwell migration assay and scratch assay were employed to determine the migration abilities of cancer cells. Fluorescence microscopy and flow cytometry were conducted to measure the intracellular reactive oxygen species (ROS) levels in cancer cells after treated with DHA. Moreover, RT-PCR and Western blot were performed to test the alteration of associated genes on mRNA and protein level in DHA treated esophageal squamous cancer cell lines, respectively. Furthermore, tumor-bearing nude mice were employed to evaluate the anticancer effect of DHA in vivo. Results: We found that hTERT was significantly upregulated in esophageal squamous cancer both from TCGA database and our cohort also. Overexpression of hTERT evidently promoted the proliferation and migration of esophageal squamous cancer cells in vitro. Moreover, DHA could significantly inhibit the proliferation and migration of esophageal cancer cell lines Eca109, KYSE150, and TE1 in vitro, and significantly down-regulate the expression of hTERT on both mRNA and protein level in a time- and dose-dependent manner as well. Further studies showed that DHA could induce intracellular ROS production in esophageal cancer cells and down-regulate SP1 expression, a transcription factor that bound to the promoter region of hTERT gene. Moreover, overexpression of SP1 evidently promoted the proliferation and migration of Eca109 and TE1 cells. Intriguingly, rescue experiments showed that inhibiting ROS by NAC alleviated the downregulation of SP1 and hTERT in cells treated with DHA. Furthermore, overexpression of SP1 or hTERT could attenuate the inhibition effect of DHA on the proliferation and migration of Eca109 cells. In tumor-bearing nude mice model, DHA significantly inhibited the growth of esophageal squamous cancer xenografts, and downregulated the expression of SP1 and hTERT protein, while no side effects were observed from heart, kidney, liver, and lung tissues by HE stain. Conclusion: hTERT plays an oncogenic role in esophageal squamous cancer and might be a therapeutic target of DHA through regulating ROS/SP1 pathway.

BAP1 promotes viability and migration of ECA109 cells through KLF5/CyclinD1/FGF-BP1.

More than 40 000 patients worldwide die from esophageal cancer annually. The 5‐year survival rate of patients is only ~ 15–20%, and thus, there is an ongoing need to improve diagnosis and treatment of esophageal cancer. Breast cancer type 1 susceptibility protein (BRCA1)‐associated protein (BAP1) is a marker of poor prognosis in several cancers, including uveal melanoma, renal cell carcinoma, cholangiocarcinoma, non‐small cell lung cancer, and colorectal cancer. BAP1 mutations are early and rare events in esophageal carcinoma, but the involvement of BAP1 in progression of esophageal carcinoma is unclear. Here, we report that cell proliferation and migration were significantly enhanced in esophageal carcinoma ECA109 cells overexpressing BAP1, while they were diminished upon BAP1 knockdown. In addition, the expression of Krüppel‐like factor 5 (KLF5), CyclinD1, and FGF‐BP1 was increased by BAP1 overexpression and decreased by BAP1 knockdown. Our data suggest that BAP1 promotes cell proliferation and migration, and enhances the expression of KLF5 and its downstream genes, including CyclinD1 and FGF‐BP1, in the esophageal carcinoma cell line ECA109.

Oesophageal squamous cell carcinoma-associated IL-33 rewires macrophage polarization towards M2 via activating ornithine decarboxylase.

BackgroundThe tumour microenvironment primarily constitutes macrophages in the form of an immunosuppressive M2 phenotype, which promotes tumour growth. Thus, the development of methodologies to rewire M2‐like tumour‐associated macrophages (TAMs) into the M1 phenotype, which inhibits tumour growth, might be a critical advancement in cancer immunotherapy research.MethodsThe expressions of IL‐33 and indicators related to macrophage polarization in oesophageal squamous cell carcinoma (ESCC) tissues and peripheral blood mononuclear cell (PBMC)–derived macrophages were determined. Inhibition of ornithine decarboxylase (ODC) with small interfering RNA was used to analyse the phenotype of macrophage polarization and polyamine secretory signals. CCK‐8, wound‐healing and Transwell assays were used to detect the proliferation and migration of ECA109 cells in vitro. The tumour xenograft assay in nude mice was used to examine the role of IL‐33 in ESCC development in vivo.ResultsThis study showed the substantially elevated IL‐33 expression in ESCC tissues compared with the normal tissues. Additionally, enhanced infiltration of M2‐like macrophages into the ESCC tumour tissue was also observed. We observed a strong correlation between the IL‐33 levels and the infiltration of M2‐like macrophages in ESCC tumours locally. Mechanistically, IL‐33 induces M2‐like macrophage polarization by activating ODC, a key enzyme that catalyses the synthesis of polyamines. Inhibition of ODC suppressed M2‐like macrophage polarization. Finally, in vivo, we confirmed that IL‐33 promotes tumour progression.ConclusionsThis study revealed an oncogenic role of IL‐33 by actively inducing M2‐like macrophage differentiation; thus, contributing to the formation of an immunosuppressive ESCC tumour microenvironment. Thus, IL‐33 could act as a novel target for cancer immunotherapies.

Effect of HMGB1 knockdown by RNA interference on cell proliferation and migration of esophageal squamous cell carcinoma after X-ray radiation

Objective To evaluate the effect of X-ray radiation on cell proliferation, migration, survival ability and cell cycle of human esophageal squamous cell carcinoma after RNA interference-mediated down-regulation of HMGB1 gene expression. Methods The expression of HMGB1 at mRNA and protein levels in the human esophageal squamous cell carcinoma cell lines ECA109 and KYSE30 was determined using RT-PCR and Western blot assays. MTS and Transwell assays were employed to examine the proliferation and migration of ECA109 and KYSE30 cell lines. The cellular survival ability in vitro was assessed by clone formation assay. The cell cycle after X-ray radiation in different groups was detected by flow cytometry. Results The expression of HMGB1 at mRNA and protein levels in ECA109 and KYSE30 cells were markedly higher in a dose-dependent and time-dependent manner in the radiation group than that in the control group (all P<0.05). MTS results demonstrated that the proliferation of ECA109 and KYSE30 cells was obviously lower at each time point after radiation than that in the group without radiation (all P<0.01). The expression of HMGB1 at mRNA and protein levels was significantly inhibited in the HMGB1 siRNA group than those in the control and NC groups (both P<0.01). The data from the clone formation assay revealed that the radiosensitivity was significantly increased after down-regulation of HMGB1 expression (P<0.01). Transwell migration assay revealed that the number of migrating cells at the fourth hour after X-ray irradiation in the HMGB1 siRNA group was significantly lower than those in the control and negative groups (both P<0.01). In the HMGB1 siRNA group, the percentage of cells at G0/G1 phase was obviously higher, whereas the percentage of S phase was significantly lower than those in the control and NC groups, and the trend was even more significant after X-ray radiation (all P<0.01). Conclusion Inhibition of HMGB1 expression by siRNA can suppress the proliferation and migration of ECA109 and KYSE30 cells and enhance the radiosensitivity by increasing the cell cycle arrest at G0/G1 stage after X-ray irradiation in vitro. Key words: HMGB1 gene; Cell proliferation and migration; Cell cycle; Radiosensitivity

Effects of Polygonatum sibiricum Polysaccharides (PSP) on Human Esophageal Squamous Cell Carcinoma (ESCC) via NF-κB Signaling Pathway

Objective. To explore the effects of different concentrations of Polygonatum sibiricum polysaccharides (PSP) on human esophageal squamous cell carcinoma (ESCC) cell line Eca109 and explore the new approach for the treatment of ESCC. Methods. Eca109 cells were divided into 5 groups, including one control group and 4 experimental groups where the concentrations of PSP used were 50, 100, 200, and 400 μg/mL. The proliferation rate of Eca109 cells in each group was measured with the CCK8 assay, and the apoptosis rate in each group was analyzed by flow cytometry; the in vitro scratch assay was used to determine the migration ability of Eca109 cells after PSP treatment; the expression levels of IL-1, IL-6, IL-10, TNF-α, and TGF-β were measured by RT-PCR, and the expression levels of TLR4 and proteins that are related to NF-κB signaling pathways were determined by Western blot. Results. PSP significantly inhibited the proliferation of Eca109 cells (p&lt;0.05) on a time- and dose-dependent manner; the apoptosis rates of Eca109 cells in experimental groups were significantly increased after 48 h of culture (p&lt;0.05); PSP significantly reduced the migration and invasion ability of Eca109 cells (p&lt;0.05); RT-PCR results showed that the expression of IL-10 in Eca109 cells increased significantly after treatment with PSP (p&lt;0.05), while the expression of IL-1, IL-6, TNF-α, and TGF-β decreased significantly (p&lt;0.05). Compared with the control group, the expression level of TLR4, NF-κB/p50, and NF-κB/p65 protein in each experimental group was significantly lower than that in the control group (p&lt;0.05). Conclusions. PSP significantly inhibited the proliferation, invasion, and migration of Eca109 cells and promoted cell apoptosis. These observed effects were probably due to the PSP’s inhibition on the NF-κB signaling pathway in Eca109 cells via the regulation of the TLR4 expression.

Matrix Metalloproteinase (MMP)-12 Promote Invasion and Migration of Esophageal Squamous Cell Carcinoma Cell by Epithelial-Mesenchymal Transition

The present study aimed to investigate the role of matrix metalloproteinase (MMP)-12 on cell migration and invasion of esophageal squamous cell carcinoma (ESCC) though epithelial-mesenchymal transition (EMT). The ESCC cell lines (ECA-109, KYSE-30, KYSE-410, KYSE-520) and normal esophageal epithelial cells (HEEC) were cultured. ECA-109 cells were then chosen to be transfected with the plasmids of MMP-12 over-expression, MMP-12 inhibitor, E-cad over-expression and empty control vectors. The protein and mRNA levels of MMP-12 were detected using western blot and qRT-PCR analysis. Transwell and wound healing assays were used to assess cell invasion and migration. Results indicated that MMP-12 was upregulated significantly in all the ESCC cell lines. Overexpression of MMP-12 increased MMP-12 expression, and the abilities of invasion and migration of ECA-109 cells. Overexpression of MMP-12 increased the expression of N-cad and vimentin, but decreased E-cad expression. Additionally, we found that cells treated with inhibitor-MMP-12 were opposite to the above results. Moreover, up-regulation of E-cad were eliminated all effects on ECA-109 cells caused by MMP-12 over-expression. In conclusion, MMP-12 promoted ECA-109 cells migration and invasion by the alteration of the EMT marker protein, which is one of its mechanisms. Therefore, MMP-12 may be a new therapeutic target for ESCC.

Experimental study on the contribution of costimulatory molecule B7-H3 in regulation of the biological function of human esophageal cancer cell line Eca-109

Objective To investigate the role of B7-H3 in the regulation of biological function of human esophageal cancer cell by using RNA interference (RNAi) methods. Methods Obtaining LV-B7-H3-siRNA and negative control LV-NC-Eca-109 by lentivirus infecting and followed by fluorescence-activated cell sorter (FACS) Aria II cell sorting system. The expression level of B7-H3 in LV-B7-H3-siRNA-Eca-109 cells and LV-NC-Eca-109 was confirmed by using real-time quantitative polymerase chain reactionB (Real-time PCR), FACS and Western blotting methods. The cell proliferation was determined by cell counting kit-8 (CCK-8) assay, and the migration and invasion was determined by wound healing assay as well as transwell assay respectively. Results We constructed the recombinant lentivirus of siRNA targeting B7-H3. On day 2 (0.31±0.03 vs. 0.36±0.02), 4 (0.51±0.04 vs. 0.60±0.04), 5 (0.60±0.02 vs. 0.70±0.03) and day 6 (0.65±0.05 vs. 0.76±0.03), the proliferation rate of LV-B7-H3-siRNA group was significantly lower than that of LV-NC control group by using CCK-8 assay (P=0.011, 0.024), respectively. The wound healing assay on Eca-109 cells in LV-B7-H3-siRNA group as well as LV-NC group showed that, the cell-free area of the LV-B7-H3-siRNA group was significantly wider than that of LV-NC group at 12 h [(38.0±28.1) vs. (467.7±30.3) pix, P=0.042], 20 h [(509.7±6.2) vs. (403.3±16.7) pix, P=0.001] and 24 h [(454.7±19.9) vs. (355.0±32.1) pix, P=0.010] respectively after drawing the scratch line on the monolayer cells. The transwell invasion assay showed that at the time point of 24 h, the number of invaded cells in the LV-B7-H3-siRNA group (258.8±29.1) cells was significantly lower than that in the LV-NC group (474.0±85.3) cells (P=0.014). Conclusion We successfully constructed the recombinant lentivirus of siRNA targeting B7-H3, and the cellular studies showed that the down-regulation of B7-H3 could suppress the biological functions such as proliferation, migration and invasion of the Eca-109 cells. Thus the over-expression of B7-H3 may involve in the malignant transformation of human esophageal cancer. The B7-H3 molecule could be developed as the potential therapeutic targets for immunotherapy of this malignancy. Key words: Co-stimulatory molecule; B7-H3; Esophageal cancer; Invasion; Metastasis

Effect of galectin3 on proliferation and migration of esophageal cancer Eca109 cells

Objective To investigate galectin3 on proliferation and migration of esophageal cancer Eca109 cells.Methods A lentiviral vector for over-expression of RNA targeting galectin3 was designed to transfect Eca109 cancer cells following plasmid-mediated transfection manual (Eca109/Gal3 cells).Inverted fluorescence microscope was used to observe the expression of EGFP.The proliferation of Eca109 cells was measured by cell counting Kit-8 assay.Eca109 cells apoptosis was determined by Annexin-V/7-AAD doublestaining.The migration capacity of Eca109 cells was determined in transwell assays.Western blot analysis was used to measure the expression of galectin3 protein.Results Galectin-3 expression was detected in Eca109 cells,with the Galectin3 expression in Eca109/Gal3 cells much more than non-transfected cells (t =14.33,P ＜ 0.05 ; t =10.28,P =0.037).Compared with non-transfected Eca109 cells,proliferation increased significantly in Eca109/Gal3 cells (t =-17.277,P ＜ 0.05 ; t =-13.4,P ＜ 0.05).Galectin3 evidently decreased in Eca109 cell apoptosis (t =3.053,P ＜ 0.05 ; t =5.446,P ＜ 0.05).Transwell migration assay showed that a greater number of Eca109/Gal3 cells crossed the artificial basement membrane compared with non-transfected Eca109 cells and negative control Eca109 cells (t =3.465,P ＜ 0.05; t =3.252,P ＜ 0.05).Conclusion Galectin3 expression is detected in transfected esophageal cancer Eca109 cells,whose overexpression can result in enhanced proliferation,migration,invasion as well as reduced apoptosis.These data indicate that in-depth research of galectin-3 may prove to be a potential molecular target for the treatment of esophageal cancer. Key words: Esophageal neoplasms ; Galectin 3 ; RNA over-expression ; Biological function

Effects of S100A 6 gene silencing on proliferation and migration of human esophagus cancer cell line Eca109

Objective To investigate the effects of S100A6gene silencing on the proliferation and migration of Eca109 human esophagus cancer cells.Methods The shRNA expression vectors were constructed using the shRNA sequences designed based on human S100A6's coding sequence,and were transfected into Eca109cells via cationic liposome.The changes of S100A6mRNA and protein in Eca109cells transfected with the recombinant vectors were detected using real-time PCR and Western blotting analysis 48hours after transfection,respectively;the proliferative curves of transfected cells were plotted using MTT assay;furthermore,the change in cellular migration ability was determined using wound healing assay. Results The eukaryotic expression vector of shRNA targeting S100A6 was successfully constructed.Real-time PCR and Western blotting analysis results showed that S100A6 was effectively silenced by liposome-mediated transfection of the recombinant shRNA vectors in Eca109cells.Compared with the untransfected cells,S100A6mRNA and protein in transfected Eca109cells were significantly decreased(P0.05,P0.01).Meanwhile,the proliferative activity of Eca109cells was significantly inhibited by S100A6silencing(P0.01).It was found that the cellular migration was also suppressed by S100A6 gene interference.Conclusion S100A6gene can be effectively silenced by shRNA expression vectors,and the silence may lead to inhibition of the proliferation and migration of Eca109cells.