Effects of cinnamaldehyde on apoptosis of oesophageal squamous cell carcinoma Eca109 cells and its mechanism / 中草药

Objective: To investigate effects of cinnamaldehyde (CA) on the proliferation and apoptosis of oesophageal squamous cell carcinoma Eca109 cells and to explore its mechanism. Methods: The effects of different concentrations (1.88, 3.75, 7.50, 15.00, 30.00 μg/mL) of CA on the proliferation of Eca109 cells were detected by MTS assay; Morphological changes were observed by phase contrast microscope; Flow cytometry was used to detect cell cycle distribution and apoptosis rate of Eca109 at different concentrations of CA; The expression of apoptosis-related protein Caspase-3/Caspase-9, pro-apoptosis protein Bax and anti-apoptosis protein Bcl-2 and Mcl-1 in ECA109 cells treated with different concentrations of CA for 24 h was detected by Western blotting. Results: After treated by CA with a concentration of 15.00, 30.00 μg/mL for 24 h, compared with the control group, the cell proliferation of Eca109 cells was significantly inhibited [(42.91 ± 2.15)%, (36.04 ± 2.97)% vs (100.00 ± 0.00)%, P < 0.05]; After treated by CA, Eca109 cells showed obvious apoptotic morphological changes; After treated with different concentrations of CA for 24 h, the proportion of cells in G0/G1 phase was decreased significantly (P < 0.05), while the proportion of cells in G2/M phase was increased significantly (P < 0.05); After treated by CA for 24 h, the apoptosis rate of Eca109 cells was increased significantly [(4.3 ± 0.11)%, (4.8 ± 0.07)%, (9.1 ± 0.13)% vs (1.0 ± 0.03)%, P < 0.05]; Compared with the control group, the fragment of protein Caspase-3 and Caspase-9 was significantly cleaved in Eca109 cells treated with different concentrations of CA for 24 h (P < 0.05); The expression levels of anti-apoptotic protein Bcl-2 and Mcl-1 were significantly decreased (P < 0.05), while the pro-apoptotic protein Bax expression was significantly up-regulated (P < 0.05). Conclusion: Cinnamaldehyde can inhibit the proliferation of oesophageal squamous cell carcinoma Eca109 cells and promote its apoptosis. The mechanism may be associated with the up-regulation of Caspase-3, Caspase-9, pro-apoptotic protein Bax and down-regulation of anti-apoptotic protein Bcl-2 and Mcl-1.

Inhibitory effect and mechanism of nitidine chloride on human esophageal cancer Eca109 cells / 中草药

Objective: To investigate the inhibitory effect of nitidine chloride (NC) on human esophageal carcinoma cell line Eca109 and the molecular mechanism of its induction. Methods: CCK-8 method was used to detect the inhibition rate of human esophageal cancer Eca109 cells with different concentrations of NC and different intervention time. According to the result of CCK-8 method, the experiment was divided into four groups, and the concentrations of NC in each group were 0, 5, 10, and 15 μmol/L, respectively, and the drug action time was 48 h. The apoptotic rate was detected by flow cytometry with Annexin V-FITC/PI double staining. The mRNA expressions of Caspase-3, Caspase-9, and Noxa were detected by qRT-PCR. The expressions of cleaved Caspase-3 and Bcl-2, p53, and Noxa protein levels were detected by Western blotting. Results: NC had inhibitory effect on Eca109 cells in a certain range of time and dose-dependent manner. Flow cytometry showed that NC at 5 μmol/L mainly induced early apoptosis (P < 0.01); NCs at 10 and 15 μmol/L mainly induced late apoptosis (P < 0.01). qRT-PCR results showed that the mRNA expression of Caspase-3, Caspase-9 and Noxa was increased with the increase of NC concentration, of which 10 and 15 μmol/L group increased significantly. The results of Western blotting showed that the protein levels of cleaved Caspase-3, p53, and Noxa were both increased with the increase of NC concentration (P < 0.01), but the increase of Noxa was not significant in 5 μmol/L group (P < 0.01). The expression of Bcl-2 protein was decreased with the increase of NC concentration, and which was significantly higher in 10 and 15 μmol/L groups (P < 0.05, P < 0.01). Conclusion: The inhibitory effect of NC on human esophageal carcinoma Eca109 cells is mainly through apoptosis. The apoptosis of NC induced of Eca109 cells is associated with increased expression of p53 and Noxa, downregulation of Bcl-2, and activation of Caspase-3.

Radiosensitizion effect of stattic on the xenograft of ECA109 esophageal cancer cells in nude mice / 中华放射医学与防护杂志

Objective To investigate the radiosensitization effect of stattic on the xenograft of esophageal squamous cell carcinoma ( ESCC ) ECA109 cells in nude mouse and explore the underlying mechanisms. Methods Male nude mice inoculated subcutaneously with ECA109 cells were used to examine the radiosensitization effect of stattic in vivo. When average volume of tumors was about 150 mm3 , mice were randomly divided into 4 groups:control group, 25 mg/kg stattic alone group, ionizing radiation (IR) (6 Gy) alone group, and 25 mg/kg stattic plus IR group. During the term of treatment, the volume of tumors was measured every 2 days. On 25 days after treatment, the mice were killed and the expressions of pSTAT3, STAT3, HIF-1α and VEGF in ECA109 xenografts were assessed by Western blot and immunohistochemistry analysis. Results The volume of tumor in nude mice was (705. 1 ± 75. 5) mm3 in stattic plus IR group, which was significantly smaller than that in IR group (113. 5 ± 101. 4) mm3 and stattic alone group(1696.5 ±100.6)mm3(t=4.35, 14.14,P<0.0). The inhibition rate was (66.1 ± 3. 2)％ in stattic plus IR group. The expression levels of pSTAT3, HIF-1α and VEGF were obviously decreased in the stattic plus IR group compared with other groups (t=17. 07, 5. 05, 3. 54, P<0. 05). Conclusions Stattic play a radiosensitization role in the xenograft of esophageal carcinoma in nude mice probably by inhibiting the signaling pathways of pSTAT3, HIF-1α and VEGF.

Tonglian Decoction () arrests the cell cycle in S-phase by targeting the nuclear factor-kappa B signal pathway in esophageal carcinoma Eca109 cells / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the anti-tumor activity and molecular mechanism of Tonglian Decoction (, TLD) on esophageal carcinoma Eca109 cells.</p><p><b>METHODS</b>Eca109 cells were treated with TLD and its separated formulae, including the clearing-heat and detoxification formula (Q), activating-blood and promoting-qi formula (H) and nourishing-yin and blood formula (Z). Cell proliferation was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay, cell morphology was observed using a microscope, the cell cycle was measured using flow cytometry and the activity of the nuclear factor-kappa B (NF-κB) signal pathway was detected by Western blot.</p><p><b>RESULTS</b>The half maximal inhibitory concentrations of TLD, Q and H were 386, 771 and 729 mg/L, respectively. TLD, Q and H significantly inhibited cell proliferation, with 69.43%, 60.84% and 61.90% of treated cells in the G phase of the cell cycle. The percentage of cells in S phase increased significantly after treatment with TLD, Q, and H compared with the control group (P<0.05), and TLD showed the strongest effect. Z had no influence on the cell cycle compared with the control group (P>0.05). Western blot detection indicated slight differences in the inhibition of the NF-κB pathway by the different formulae. TLD formula strongly inhibited IKKβ, NF-κB, interleukin-6 and tumor necrosis factor-α expression compared with the control group.</p><p><b>CONCLUSIONS</b>TLD inhibited Eca109 cell proliferation by arresting cells in S phase. The possible mechanism might be related to inhibiting the NF-κB transduction cascade. The combination of the herbs found in the three separate formulae, H, Q and Z, work synergistically in TLD to produce the inhibitory effects of TLD treatment on Eca109 proliferation.</p>

Chemical constituents of ethanol extracts from Daphne altaica pall and their pro-apoptotic role in cancer cell / 天津医药

Objective To investigate the chemical constituents of the ethanol extract of Daphne altaica Pall (DA-Et), to determine total phenolic content in DA-Et and to study the effect of DA-Et on cell apoptosis in Eca-109 cells. Meth?ods Different chemical assays were performed to detect chemical constituents of DA-Et. The content of total phenolics ,ex?pressed as gallic acid equivalents,was measured by Folin-Ciocalteu assay; Cell apoptosis induced by DA-Et was detected by AnnexinV-FITC/PI flow cytometry. Results Flavonoids,coumarins,sugars,alkaloids and phenolics in DA-Et. The contents of total phenolics in DA-Et was quantified as 159.78 mg/g. Average recovery rate of this method ranged from 99.4% to 108.3% and the precision relative standard deviation was 2.04%. Apoptosis rate of Esophageal cancer cell line Eca-109 induced by DA-Et was detected as 29.633%±1.779%(n=3). Conclusion Our study provide guidance for the im?provement of DA-Et quality, its appropriate application and further research of its activity.

Autophagy inhibition enhances radiosensitivity of esophageal squamous carcinoma Eca-109 cells / 中华放射医学与防护杂志

Objective To investigate the role of autophagy in radiation-induced death process of human esophageal squamous carcinoma Eca-109 cells.Methods Esophageal carcinoma cell line Eca-109 was divided into 6 groups of control,5 mmol/L 3-Methyladenine treatment,10 mmol/L treatment,6 Gy irradiation,irradiation + 5 mmol/L drug,and irradiation + 10 mmol/L drug.Some cells were transferred with GFP-LC3 plasmid and the changes of autophagosome were obserred.After each treatment,the expression of autophagy marker LC3B was measured by Western Blot,cell viability was detected by MTT,morphological characteristics of apoptosis cells were stained with a fluorescein of Hoechst 33342 and the percentage of apoptotic cells and cell cycle distribution were measured by flow cytometry.Clonogenic survival were used to evaluate the cell radiosensitivity.Results Autophagy level was increased after radiation,and the LC3B Ⅱ expression and LC3B Ⅱ/LC3B Ⅰ ratio were significantly decreased by autophagy inhibitor 3-Methyladenine (F =25.64,P ＜ 0.05).The number of autophagosome fluorescent foci were significantly increased in the GFP-LC3 transfected cells after radiation,but reduced by 3-Methyladenine (F =127.36,P ＜ 0.05).Compared with radiation alone group,autophagy inhibition combined with radiation significantly decreased cell viability (F =129.54,P ＜ 0.05) and colony formation,increased apoptosis and the percentage of G2/M-phase cells.Conclusions 3-Methyladenine enhances the radiosensitivity of esophageal squamous carcinoma Eca-109 cells,suggesting that inhibition of autophagy could be used as an adjuvant treatment of radiotherapy in esophageal squamous carcinoma.

Effect of DEC1 gene over-expression on proliferation and invasion abili-ties of human esophageal cancer ECA109 cells / 中国病理生理杂志

[ ABSTRACT] AIM:To investigate the effect of DEC1 gene over-expression on the proliferation and invasion abili-ties of human esophageal cancer ECA109 cells.METHODS: ECA109 cells were transfected with plasmid pcDNA3.1 (-)/DEC1 (DEC1 group) or pcDNA3.1 (-) (vector group).The mRNA and protein levels of DEC1, cyclin D1 and MMP-9 were evaluated by real-time PCR and Western blot, respectively.The effects of DEC1 over-expression on the prolif-eration and invasion abilities of the ECA109 cells were evaluated by CCK-8 assay, colony formation assay and Transwell test respectively.RESULTS:The DEC1 expression level in ECA109 cells in DEC1 group was significantly higher than that in vector group (P<0.01), but the levels of MMP9 and cyclin D1 expression were opposite (P<0.01).However, both the proliferation and invasion abilities of ECA109 cells in DEC1 groups decreased significantly as compared with those in vector group (P<0.05).CONCLUSION:The over-expression of DEC1 significantly inhibits the proliferation and invasion of ECA109 cells, which may be involved in the expression of cyclin D1 and MMP9.

Swainsonine promotes apoptosis in human oesophageal squamous cell carcinoma cells in vitro and in vivo through activation of mitochondrial pathway.

Swainsonine, a natural indolizidine alkaloid, has been reported to have antitumour effects, and can induce apoptosis in human gastric and lung cancer cells. In the present study, we evaluated the antitumour effects of swainsonine on several oesophageal squamous cell carcinoma cells and investigated relative molecular mechanisms. Swainsonine treatment inhibited the growth of Eca-109, TE-1 and TE-10 cells in a concentration-dependent manner as measured by MTT assay. Morphological observation, DNA laddering detection and flow cytometry analysis demonstrated that swainsonine treatment induced Eca-109 cell apoptosis in vitro. Further results showed that swainsonine treatment up-regulated Bax, downregulated Bcl-2 expression, triggered Bax translocation to mitochondria, destructed mitochondria integrity and activated mitochondria-mediated apoptotic pathway, followed by the release of cytochrome c, which in turn activated caspase-9 and caspase-3, promoted the cleavage of PARP, resulting in Eca-109 cell apoptosis. Moreover, swainsonine treatment inhibited Bcl-2 expression, promoted Bax translocation, cytochrome c release and caspase-3 activation in xenograft tumour cells, resulting in a significant decrease of tumour volume and tumour weight in the swainsoninetreated xenograft mice groups compared with that in the control group. Taken together, this study demonstrated that swainsonine inhibited Eca-109 cells growth through activation of mitochondria-mediated caspase-dependent pathway.

Effect of JAK2 inhibitor on proliferation,apoptosis and COX-2 expression of esophageal carcinoma Eca-109 cell line / 基础医学与临床

Objective To investigate the relationship of STAT3 signal transduction pathway with proliferation,apoptosis and COX-2 expression of human esophageal carcinoma Eca-109 cell lines.Methods Eca-109 cells were treated with selective JAK2 inhibitor,AG490.MTT assay was used to detect the proliferation of Eca-109 cells,apoptosis was detected by flow cytometry,agarose gel electrophoresis of DNA and transmission electron micrograph(TEM).The expression of JAK2、p-JAK2、p-Stat3 and COX-2 was examined by Western blot.RT-PCR was performed to detect the levels of COX-2 mRNA expression.Results AG490 significantly inhibited the growth of human Eca-109 cells in a dose and time-dependent manner and induced apoptosis.AG490 inhibited the expressions of JAK2/STAT3 signal transduction pathway protein and down-regulated the expressions of p-JAK2 and p-Stat3(P

Construction of signal peptide-canstatin expression vector and its secretable expression in Eca-109 cells / 中国病理生理杂志

AIM: To construct signal peptide-canstatin expression vector pEGFP-C1-SP-Can and express secretable mouse canstatin fusion protein in Eca-109 cells.METHODS: Site-directed mutagenesis was used in amplifying the signal peptide of murine plasminogen to construct the plasmid pEGFP-C1-SP.The cDNA of mouse canstatin,obtained from a cloning vector pMD18T-Can by PCR,was inserted into pEGFP-C1-SP to construct a secretable expression vector pEGFP-C1-SP-Can.Constructed plasmid pEGFP-C1-SP-Can was transiently transfected into Eca-109 cells via lipofectamine,and subsequently its secretable expression in the medium of cultured Eca-109 was observed by Western blotting.RESULTS: DNA sequencing and restriction enzyme analysis attested the validity of the constructed plasmids pEGFP-C1-SP and pEGFP-C1-SP-Can.EGFPcanstatin fusion protein was proved to be secretably expressed in Eca-109 by Western blotting.CONCLUSION: It is concluded that the constructed vector pEGFP-C1-SP-Can is valid and capable of expression in Eca-109,these findings provide a basis for testing the function of mouse canstatin and its application in gene therapy.