Effect of different expression levels of USP28 on apoptosis and related protein expression in esophageal cancer cell ECA109 after irradiation / 中华放射肿瘤学杂志

Objective To observe the effect of different expression levels of USP28 on the radiosensitivity of ECA109 cells by gene transfection method,aiming to provide theoretical basis for comprehensive treatment of esophageal cancer.Methods The expression levels of USP28 and c-Myc in the esophageal epithelial cells Het-1A,ECA109 and ECA109R were quantitatively measured by qRT-PCR.The specific siRNA sequences were designed according to the USP28 and c-Myc genes.The pcDNA-USP28 and pcDNA-c-Myc plasmids were constructed.The esophageal cancer cell ECA109 was transfected with Lipofectamine 2000 to observe the transfection effect and related protein expression.ECA109 and ECA109R cells were exposed to 6 Gy X-ray radiation.The cell apoptosis in each group was detected by flow cytometry.The radiosensitivity was evaluated by clone formation assay.Results The expression levels of USP28 and c-Myc in ECA109 were significantly higher than those in Het-1A (both P＜0.05),and the expression levels of USP28 and c-Myc in ECA109R were remarkably higher than those in ECA109(both P＜0.05).The pcDNA-USP28 and pcDNA-c-Myc recombinant plasmids were successfully constructed.Compared with the negative control group,the expression of USP28 at the protein and mRNA levels in the si-USP28 group was significantly down-regulated,whereas those in the pcDNA-USP28 group were remarkably up-regulated.Similar results were obtained in terms of c-Myc.Compared with the control group,the expression level of c-Myc protein was significantly up-regulated in the pcDNA-USP28 group,whereas considerably down-regulated in the si-USP28 group.After 6 Gy irradiation,the apoptosis rate and radiosensitivity of ECA109 cells were significantly declined.The apoptosis rate and radiosensitivity of ECA109R cells were increased in the si-USP28 group.Conclusions The expression of USP28 protein is closely correlated with the radiosensitivity of esophageal cancer cells.The underlying mechanism may be related to the regulation of c-Myc expression by USP28.

Effect of different expression levels of USP28 on apoptosis and related protein expression in esophageal cancer cell ECA109 after irradiation / 中华放射肿瘤学杂志

Objective@#To observe the effect of different expression levels of USP28 on the radiosensitivity of ECA109 cells by gene transfection method, aiming to provide theoretical basis for comprehensive treatment of esophageal cancer.@*Methods@#The expression levels of USP28 and c-Myc in the esophageal epithelial cells Het-1A, ECA109 and ECA109R were quantitatively measured by qRT-PCR. The specific siRNA sequences were designed according to the USP28 and c-Myc genes. The pcDNA-USP28 and pcDNA-c-Myc plasmids were constructed. The esophageal cancer cell ECA109 was transfected with Lipofectamine 2000 to observe the transfection effect and related protein expression. ECA109 and ECA109R cells were exposed to 6 Gy X-ray radiation. The cell apoptosis in each group was detected by flow cytometry. The radiosensitivity was evaluated by clone formation assay.@*Results@#The expression levels of USP28 and c-Myc in ECA109 were significantly higher than those in Het-1A (both P<0.05), and the expression levels of USP28 and c-Myc in ECA109R were remarkably higher than those in ECA109(both P<0.05). The pcDNA-USP28 and pcDNA-c-Myc recombinant plasmids were successfully constructed. Compared with the negative control group, the expression of USP28 at the protein and mRNA levels in the si-USP28 group was significantly down-regulated, whereas those in the pcDNA-USP28 group were remarkably up-regulated. Similar results were obtained in terms of c-Myc. Compared with the control group, the expression level of c-Myc protein was significantly up-regulated in the pcDNA-USP28 group, whereas considerably down-regulated in the si-USP28 group. After 6 Gy irradiation, the apoptosis rate and radiosensitivity of ECA109 cells were significantly declined. The apoptosis rate and radiosensitivity of ECA109R cells were increased in the si-USP28 group.@*Conclusions@#The expression of USP28 protein is closely correlated with the radiosensitivity of esophageal cancer cells. The underlying mechanism may be related to the regulation of c-Myc expression by USP28.

The effect of RNF2 knockdown on the radiosensitivity in transplanted tumor of nude mice of esophageal carcinoma in vivo / 中华放射肿瘤学杂志

Objective To evaluate the effect of RNF2 gene knockdown in ECA109 cells on the radiosensitivity to esophageal cancer cell xenograft in nude mice. Methods Thirty-six male BALB/c/nu nude mice were randomly divided into 6 groups: control group, control+ irradiation group, NC group, NC+irradiation group, RNF2 shRNA group and RNF2 shRNA+ irradiation group. The nude mouse models with transplanted tumors were established by subcutaneous inoculation of EAC109 cells and given with irradiation at a dose of 3 Gy for 5 times. The longest ( a) and shortest ( b) diameters of the transplanted tumor were measured every 2 to 3 day since the fourteenth day after inoculation. The time of tumor formation was recorded. The tumor volume was calculated according to the formula ( ab2/2 ) . The growth curve was delineated. Three nude mice were sacrificed in each group at 24 h after the initial irradiation. The expression of RNF2 at the mRNA and protein levels in transplanted tumor tissues was measured by qRT-PCR and immunohistochemistry, respectively. The growth and tumor volume of the other nude mice in each group were observed. The cell apoptosis of transplanted tumor tissues was detected by TUNEL assay. The expression of Bcl-2 and Bax at the mRNA and protein levels in transplantated tumor tissues was quantitatively measured by qRT-PCR and immunohistochemistry, respectively. Results The tumor growth rate was the highest in the control and NC groups. The knockdown of RNF2 reduced the growth rate of xenografts and the tumor growth rate was the slowest in the RNF2 shRNA+ irradiation group ( P<0.05) . TUNEL assay revealed that the cell apoptosis rates in all groups were significantly increased after irradiation ( all P<0.05) . Before and after irradiation, the apoptosis rate in the RNF2 shRNA group was markedly higher than those in the control and NC groups ( both P<0.05) . Prior to irradiation, the expression levels of RNF2 mRNA and protein in the RNF2 shRNA group were significantly lower compared with those in the control and NC groups ( all P<0.05) , and the tendency became more significant after irradiation. Compared with the control and NC groups, the expression levels of Bcl-2 mRNA and protein were significantly down-regulated in the RNF2 shRNA group before and after irradiation ( all P<0.05) , whereas those of Bax mRNA and protein were considerably up-regulated ( all P<0.05 ) . Conclusions In vivo experiment demonstrates that RNF2 knockdown effectively increases the radiosensitivity of esophageal carcinoma EAC109 cells in nude mouse models with transplanted tumors, which is intimately associated with inducing the cell apoptosis.

Effect of lentivirus-mediated expression of RNF2 gene on the proliferation and migration of esophageal carcinoma cells following X-ray radiotherapy / 中华放射肿瘤学杂志

Objective To examine the effect of X-ray radiotherapy on cell proliferation, migration, apoptosis, and cell cycle of human esophageal carcinoma ECA-109 cells following RNA interference (RNAi)-mediated downregulation of RNF2 gene expression.Methods The level of RNF2 mRNA expression in the human esophageal carcinoma cell line ECA-109 was determined using RT-PCR.Cell proliferation of ECA-109 was measured by MTT assay, and the changes in RNF2 protein expression in ECA-109-R cells were determined using Western blot.The changes in cell cycle and cell apoptosis at different time points following radiation were analyzed by flow cytometry, and the effect of transduction on cell migration was examined using Transwell migration assay.Data were subjected to an analysis of variance with repeated measurement design.Results The mean mRNA and protein levels of RNF2 in ECA-109 cells were significantly increased in a dose-dependent manner in the radiation group than in the control group (P0.05).The Transwell migration assay showed that the number of migrating cells following 3.5 h of radiation was significantly lower in the ECA-109-R group than in the ECA-109 and ECA-109-N groups (P<0.01).The percentage of G2/M phase cells in each group was significantly lower following 6 Gy radiation compared to that in the corresponding untreated group (P<0.01), and the percentage of G2/M phase cells was significantly lower in the ECA-109-R group than in the ECA-109 and ECA-109-N groups (P<0.05).Furthermore,cell apoptosis following radiation was also significantly higher in the ECA-109-R group than in the ECA-109 and ECA-109-N groups (P<0.01).Conclusions RNAi-mediated downregulation of RNF2 expression in esophageal carcinoma cells can reduce cell proliferation and cell migration, rescue post-radiation G2/M cell cycle arrest, promote cell apoptosis,and increase radiosensitivity.

Effect of oridonin on the gene expression of human esophageal carcinoma cell SHEEC and the target gene screening / 肿瘤研究与临床

Objective To detect the effect of oridonin(ORI)on the gene expression of human esophageal carcinoma cell SHEEC and to screen the tumor cell apoptosis target genes.Methods The gene expression of human esophageal carcinoma cell SHEEC without and with ORI induction for 1 hours and 8 hours were detected with microarray technique,respectively.The differentially expressed genes were identified and verified with fluorecent quantitative PCR.Results A total of 1011 genes showed up or down regulation more than twice after ORI induction(including 280 genes after 1 hour and 731 genes after 8 hours induction respectively).In these genes,17 genes with the top extent of up or down regulation were identified,which were involved in the cell signal transduction,transcription regulation,and cell apoptosis.These 17 differentially expressed genes were verified with real-time PCR,and 12 genes were statistically significant.Conclusion In the 12 differentially expressed genes with statistically significance,there may have tumor cell apoptosis target genes induced by ORI through mitochondrion route.

ERCC2 expression in siRNA interference of esophageal cancer cells and correlation with paclitaxel sensitivity / 中国癌症杂志

Background and purpose: ERCC2 gene silence by siRNA interference was observed in esophageal cancer cell line and it could change cell sensitivity to taxol. This study was to investigate the biological mechanism of paclitaxel-resistance in esophageal cancer cells. Methods: ERCC2 targeting siRNA (si-ERCC2) has been synthesized. The constructor were transfected into ERCC2 cell lines high-KYSE150(bigh expression of ERCC2) through lipofectamine. RT-PCR and flow cytometry (FCM) were used to detect the ERCC2 mRNA and protein expression levels. MTr assay was used to estimate the paclitaxel sensitivity of the cells. Results: In si-ERCC2 group, ERCC2 could not be detected and ERCC2 protein expression were reduced by 31.2%, 51.6% and 60.0%, respectively, 24,48,72 b after transfection. Paclitaxel IC_(50) value for si-ERCC2 group was 6.32±0.87 μg/mL, lower than the control group(49%). Conclusion: siRNA could successfully silence the target gene ERCC2 at the level of transcription and translation of the gene, the reduction of ERCC2 expression may reverse taxol resistance of the cells.

Study on 3-nminobenzamide enhancing the radiosensitivity of human esophageal carcinoma cell strain in vitro / 中华放射医学与防护杂志

Objective To evaluate the ability of 3-AB to sensitize the human esophageal carcinoma cell strain (CaEs-17) to radiation in v/tro and its mechanisms. Methods CaEs-17 cells were treated with 3-AB at 0, 2.5, 7.5 mmol/L and given irradiation O, 3, 6, 9, 12 Gy. 3-AB concentration in each group was made dose-survival curve using multi-target single-hit maiths model by clonogenie assay. MTT assay was performed to observe the survival of irradiated cells.comet assay and metaphase chromosome analysis were used to measure the DNA damage degree and chromosome aberration of CaEs-17 cell after 3-AB treatment and irradiation. Results Cell survival experiments showed SER of 1.21, 1.52 for 2.5 mmol/L, 7.5 mmol/L 3-AB respectively using multi-target single-hit maths model. The survival fraction of irradiated CaEs-17 cell was decreased after 3-AB treatment. DNA damage and the chromatid breakage number of irradiated CaEs-17 cells were increased after 3-AB treatment. Conclusions 3-AB, a PARP inhibitor, can enhance the radiosensitivity of human esophageal carcinoma cell strain (CaEs-17). DNA damage repair inhibition by 3-AB might be one of the mechanisms.

Effects of extracellular ATP on apoptosis of human esophageal carcinoma Eca-109 and hepatoma SMMC-7721 cells / 基础医学与临床

Objective To study the effects of adenosine triphosphate (ATP) on proliferation of human squamous esophageal carcinoma Eca-109 and hepatoma SMMC-7721 cells in vitro and the underlying mechanism. MethodsMTT assay was used to determine the proliferation of tumor cells. The AO/EB double stained cells were observed under fluorescence microscope. The effects of ATP on the cell cycle, apoptotic rate and apoptosis-related protein were detected by flow cytometry. Results ATP showed inhibitory effects on Eca-109 and SMMC-7721 cells at the concentration of 0.01～0.3 mmol/L. Exposure to 0.3 mmol/L ATP for 72 h, some of SMMC-7721 cells displayed morphological changes of apoptosis, but Eca-109 cells did not show the characteristics of apoptosis markedly. There was no significant change in the apoptotic rate and apoptosis-related protein of the two tumor cell lines treated with ATP 0.03, 0.1 and 0.3 mmol/L for 72 h respectively. The proportion of Eca-109 cells in G0/G1-phase of cellcycle was significantly increased, meanwhile the proportion of Eca-109 cells in S-phase and proliferation index value was significantly decreased by treatment with 0.3 mmol/L ATP. Conclusion ATP inhibited Eca-109 cell proliferation by changing the distribution of cell cycle phase, and its mechanism might not related to apoptosis, but for SMMC-7721 cell, the inhibition of cell proliferation induced by ATP was not related to the change in cell cycle.

Induction of apoptosis and influence of HIF-1? expression on esophageal carcinoma cell ECa109 by soybean isoflavone during hypoxia / 第三军医大学学报

Objective To study the mechanisms and effect of soybean isoflavone on esophageal carcinoma cell ECa109 during the normoxia and hypoxia. Methods The environment of hypoxia was established by GasPak method. The ECa109 cells were assigned into normal control group, soybean isoflavones group, hypoxia group, and soybean isoflavones plus hypoxia group. The effect of soybean isoflavones was determined by MTT, and FCM was used for detecting the apoptosis and cell cycle of ECa109. Electron microscope was used to observe the ultrastructural changes of ECa109 induced by soybean isoflavone. Immunohistochemistry was used to detect the changes of HIF-1? and Fas. Results Soybean isoflavone could inhibit significantly the growth of ECa109 cells in a concentration-dependent manner, and arrest the cells in G_2/M phase during normoxia and hypoxia. The inhibitory effect was elevated significantly during hypoxia, and the cell apoptosis and necrosis were observed by electron microscope. The Fas expression was elevated by soybean isoflavone during the normoxia and hypoxia, and the HIF-1? expression was down-regulated during hypoxia. Conclusion Soybean isoflavone can inhibit ECa109 cells growth by delaying the progress of cell cycle, up-regulating the Fas expression. Soybean isoflavone can inhibit the expression of HIF-1? that was increased during hypoxia, which may be the mechanism that the inhibitory effect was enhanced significantly during hypoxia.

Effects and Mechanisms of Ursolic Acid on Inducing Apoptosis of Human Esophageal Carcinoma Cell Line Eca-109 / 中药新药与临床药理

Objective To investigate the effects and possible mechanisms of ursolic acid (UA) on inhibiting proliferation of human esophageal carcinoma cell Eca-109 and inducing its apoptosis. Methods Cell proliferation was determined by MTT assay. Electron microscope was used to observe the ultrastructural changes of Eca-l09 induced by UA. Cell cycle and apoptotic rate were analyzed by flow cytometry (FCM),and the expression of P27kip1,Bcl-2 and Bax were detected by Western blot method. Results UA could significantly inhibit the growth of Eca-109 cells(P

Experimental study of cancer vaccine prepared by fusions of esophageal carcinoma cell line with dendritic cell derived from cord blood / 中国实用内科杂志

Objective To establish the cancer vaccine (EC109-DC) prepared by fusions of esophageal carcinoma cell line with dendritic cell derived from cord blood and study the biological characteristics and antitumor immunity.Methods CD 34 + hematopoietic stem cells were isolated from cord blood using CD 34 + Progenitor Cell Isolation Kit by magnetic cell sorting system (MACS);expended the cells as DC,and then fused with EC109 by PEG-3600;selected the fusion cells by MACS;observed the phenotypes and proliferation by flow cytometry and technique of cell culture in vitro. Lymphocytes proliferation reaction and the CTL cytotoxicity were examined with MTT.Results EC109-DC could proliferate slowly in vitro and highly expressed CD80,CD83 and CD86.Lymphocytes proliferation reaction and the specific cytotoxicity against EC109 induced by EC109-DC cells were significantly higher than the control groups (P

Role of transcription factors Sp1 and Sp3 in the expressional regulation of ezrin in esophageal carcinoma cells / 中国免疫学杂志

Objective:To identify the role of transcription factors Sp1 and Sp3 in the expressional regulation of ezrin in human esophageal carcinoma cells.Methods:Esophageal carcinoma EC109 cells were transfected with expressing vectors CMV-Sp1 or CMV-Sp3,and the effect of Sp1 and Sp3 over-expression on ezrin mRNA and protein expression was determined by real time RT-PCR and Western blot analysis.Furthermore,EC109 cells were cotransfected with the ezrin promoter-directed luciferase reporter vector and control vector pRL-TK along with transcription factor expression vector.The roles of Sp1 and Sp3 in ezrin promoter activation and whether this activation occurred through the Sp1 binding site,-75/-69,were analyzed by dual-luciferase reporter assay system.Results:Over-expression of transcription factors Sp1 and Sp3 significantly increased the expression of ezrin mRNA and protein and the ezrin promoter activity in EC109 cells.Sp1 and Sp3 enhanced the promoter activity through different binding sites and only Sp1 did that through the-75/-69 site.Conclusion:Sp1 and Sp3 can regulate ezrin expression in EC109 cells.