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Objective To investigate the effects of inhibition of MDC1 protein expression on xenografted tumors in nude mice,and to observe the histopathological and cellular changes in nude mice.Methods Three pairs of effective and control short hairpin RNA targeting MDC1 mRNA were designed and cloned into the pSIH1-H1-copGFP vector.Real-time PCR and Western blot were used to determine the mRNA and protein expression of MDC1.After selection by copGFP reporter gene,cells were divided into negative transfection group (ECA109-N) and MDC1 transfection group (ECA109-M).The transfected cells were injected into nude mice.The mice were divided into ECA109 group,ECA109-N group,and ECA109-M group.Each group was divided into irradiation subgroup and non-irradiation subgroup.The changes in tumor size after irradiation were evaluated in each group.Western blot was used to measure the expression of CHK1,CHK2,and CHK2T68 in xenografted tumors.Flow cytometry was used to analyze the cell cycle distribution and apoptosis of tumor cells in nude mice.The variance analysis was used to compare the mean of multiple groups,and the SNK-q test was used in the two two groups.Results The pMDC1-shRNA plasmid was successfully constructed and used to transfect ECA109 cells.ECA109-M cells were obtained by stable transfection with the recombinant plasmid.All inoculated nude mice survived with visible xenografted tumors at the underside of the paw in about one week.There was no swelling and wound in inoculation sites.There was no significant difference in tumor size between different groups (P＞0.05).The tumor growth in the ECA109 group and the ECA109-N group significantly slowed down after irradiation with a dose of 15 Gy (P＜0.05).Compared with the other two groups,the ECA109-M group had a significant smaller tumor size,significantly slower relative tumor growth,and significantly higher growth inhibition (all P＜0.05).The q value of the ECA109-M group was 1.36.In the ECA109-M group,there were no significant changes in the protein expression of CHK1 and CHK2 after irradiation (P＞ 0.05);however,the phosphorylation of CHK2T68 protein was significantly reduced after irradiation (P＜0.05).There were no significant differences in cell cycle distribution or the proportion of apoptotic cells in tumor tissue between the three groups (P＞0.05).Conclusions Inhibition of MDC1 protein expression by RNA interference can effectively inhibit the growth of xenografted tumors after irradiation in the nude mice by increasing their radiosensitivity.
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Objective:To investigate the effects of epidermal growth factor (EGF)on cell cycle and cell cycle-related regulatory factors of human esophageal squamous cell carcinoma (ESCC) cell line Eca109.Methods: Serum starved Eca109 cells were treated with 20 ng/ml recombinant human EGF(rhEGF)for 24 h.The cell cycle phase distribution was detected by flow cytometry.The mRNA and protein expression levels of p21CIP1/WAF1(p21) and p27KIP1(p27) were detected by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)and Western blot,respectively.Results: The proportions of G1 phase cells in EGF group and control group were ( 54.90 ±0.82 )% and ( 65.94 ±0.74 )%.The mRNA and protein expression levels of p 21 in EGF group was significantly higher ,and p27 was significantly lower than that in control group ( P<0.01 ) .Conclusion: EGF facilitates G1-S phase transition,and promotes the proliferation of Eca 109 cells,which may be associated with the up-regulation of p21 and down-regulation of p27.
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Objective To construct a small interfering RNA (siRNA) vector targeting ΔNp63α and investigate ΔNp63α gene interference on the proliferation and apoptosis of human esophageal squamous carcinoma Eca109 cell line. Methods Adeno-associated virus (AAV)-ΔNp63αshRNA driven by H1 promoter was constructed and was used to infect Eca109 cells. AAV-Null and normal cell lines were utilized in the control group and blank control group, respectively. The influence of siRNA interference of ΔNp6α expression on the growth, proliferation, tumorigenic efficiency and apoptosis of Eca109 cells were analyzed in vitro and in vivo. Results Compared with the two control groups, the specific siRNA targeting ΔNp63α gene significantly down-regulated the expression of ΔNp63α protein levels in Eca109 cells (all P<0.05). The growth of Eca109 cells infected with AAV-ΔNp63αshRNA was significantly lower than those in the two control groups (all P<0.05). Cell cycle analysis showed the proliferation index (PI) of AAV-ΔNp63αshRNA infected cell line was significantly lower compared with the two control groups (all P<0.01). In vivo experiment exhibited that AAV-ΔNp63αshRNA infected cells resulted in a lower tumor weight in nude mice compared with the cells in the two control groups (all P<0.05). In addition, the apoptosis index (AI) of AAV-ΔNp63αshRNA infected cells were significantly higher than those of the other cell lines (P<0.05). Conclusion AAV- mediated expression of shRNA can significantly reduce ΔNp63α expression in Eca109 cells, slowing down the proliferation, promoting the apoptosis, and subsequently inhibiting the growth of tumor.
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Objective To study the change of DNA polymerase beta and XRCC1's expression during malignant celI differentiation.Methods The Eca-109 cells were divided inm 2 groups:differentiation group which cultured with 8-Br-cAMP and control group.The 2 groups cells were cultured 48h simultaneously.The immunocytochemistry was performed to detect the expression of DNA polymerase beta and XRCC1.Results Compared with control group,the expression of DNA polymerase beta and XRCC1 was decreased simultaneously(P＜0.05).Conclusion The differentiation agent can down-regulate the expression of DNA polymerase beta and XRCC1,suggesting that overexpressed DNA polymerase beta and XRCC1 maybe result in mutator phenotype.
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Aim To study the effects of selective cyclooxygenase-2 (COX-2) inhibitor,nimesulide,on COX-2 expression, cell proliferation and apoptosis of human esophageal carcinoma Eca-109 cell lines. Methods MTT assay was used to observe the proliferative effect;COX-2 mRNA expression was evaluated with RT-PCR; COX-2 protein expression,cell cycle and apoptosis were analyzed with flow cytometry;microscope and agarose gel electrophoresis of DNA were also used to observe the apoptosis. Results Nimesulide significantly inhibited the proliferation of Eca-109 cell lines in a time and dose-depenent fashion, down-regulated the expression of COX-2 mRNA and COX-2 protein in a dose-dependent fashion;nimesulide also decreased the proliferation index and the proportion of cells in S phase, meanwhile increased the proportion of cells in G_0/G_1 phase and induced apoptosis. Conclusion COX-2 selective inhibitor nimesulide inhibits proliferation,induces apoptosis and cell cycle arrest of human esophageal cells via down-regulation of COX-2 expression.
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Objective To investigate the in vitro inhibitory effect of LIGHT on human esophageal carcinoma cells. Methods LIGHT Fc expression vector was transfected into human esophageal squamous carcinoma cell line, Eca109. The inhibitory effect of LIGHT gene on cell growth was detected by MTT and cell growth curve. The expressions of LT?R and HVEM were detected by RT PCR. Results Expression of LIGHT Fc gene could inhibit Eca109 cell proliferation. The growth curve of Eca109/LIGHT was significantly lower than that of the control group in the culture medium containing 1% FCS. MTT test showed that there was significant difference in cell viability between Eca109/LIGHT and the control group ( P
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Aim To investigate the regulatory effect of p38MAPK signal pathway on the apoptosis of human esophageal cancer cells induced by valdecoxib.Methods The tumor cells were inoculated in the dose of 1?107?L-1.After 6 h,the cells were divided into control group,solution group,400,200,100,50,25 ?mol?L-1 valdecoxib group and various concentration valdecoxib+SB203580 group,cultured for 72 h.FCM and DNA Ladder were used to detect the apoptosis of Eca109 cells.p38 mRNA expression was detected by RT-PCR.The expression of p-p38MAPK protein was detected by immunohistochemical staining and FCM.Results ① Valdecoxib could increased the apoptosis rate of Eca109 cell in concentration-dependent fashion.Apoptosis rate was increased to 48.46% in 400 ?mol?L-1 valdecoxib group at the incubation time of 72 h.DNA ladder was the most recognized marker of apoptosis,and there was obvious DNA ladder in valdecoxib treated group,especially in 400 ?mol?L-1 group.② Valdecoxib could increase the expression of p38MAPK,while SB203580 could inhibit the over-expression induced by valdecoxib,at the same time,the apoptosis rate was been decreased.③ The expression of p38MAPK protein was positively correlated with the apoptotic rate(r=0.822,P=0.000).Conclusions Valdecoxib could activate p38MAPK pathway,thus induce the apoptosis of Eca109 cells,which may be one of the mechanisms for the inhibition of cell growth by valdecoxib.
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Aim To investigate the inhibitory effect of paeonol (Pae) on the human esophageal cancer cell line Eca-109 in vitro and in vivo and its effect on apoptosis.Methods Cytotoxic effect of Pae on Eca-109 cells cultured in vitro was measured by MTT assay.Anti-tumor activity was performed on BALB/c nude mice xenografts model.The morphologic changes of tumor tissue were observed under light microscope and transmission electron microscope.The apoptosis index was assessed by TUNEL.Results Pae had significant in- hibitory effect on the proliferation of Eca-109 cells,and the IC50 value was 0.342 mmol?L-1.In the model of human esophageal cancer xenografts in BALB/c nude mice,the inhibitory rates of Pae group (25、50、100、200 mg?kg-1) were 10.67%、23.54%、27.91% and 34.46% respectively.In vivo administration of Pae 100 mg?kg-1 combined with cisplatin 5 mg?kg-1 resulted in a significant inhibition of Eca-109 tumor growth with the inhibitory rate of 77.91%,compared with cisplatin used alone (58.71%).The more apoptotic tumor cells could be seen under light microscope in every theraperutic groups than those in control.Changes of ultrastructure of tumor cells including concentration and side accumulation of the nuclear chromatin,and the fragmentation of the nuclear was observed under transmission electronic microscope.Apoptosis body was also found.The apoptosis indexes of every theraperutic groups were significantly different from the control.Conclusion Pae can inhibit the cell growth and induce apoptosis in human esophageal cancer cell line Eca-109 in vitro and in vivo.
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Aim To investigate the effect and mechanism of valdecoxib on the apoptosis of human esophageal cancer cells.Methods Flow cytometry was used to observe the effect of valdecoxib on apoptosis and the cell cycle distribution of Eca109 cells.Transmission electron microscope was further used to detect the cell apoptosis.The content of LDH was examined using LDH kit.The expressions of p-p38MAPK,Fas and FasL protein were detected using flow cytometry.Results Valdecoxib of 25～400 ?mol?L~(-1) significantly induced the apoptosis of Eca109 cell line,and the rate of apoptosis was increased from(2.95?0.83)% to(48.46?0.73)%,50～400 ?mol?L~(-1) valdecoxib also decreased the proliferation index and the proportion of cells in the S phase,increased the proportion of cells in the G_0/G_1 phase,but had no effect on the proportion of cells in the G_2/M phase.Compared with those in Eca109 cells cultured in the medium with solvent,the expression of p-p38MAPK,Fas and FasL was higher in the Eca109 cells exposed to valdecoxib in a dose-dependent manner in 72 h.Conclusion Valdecoxib can induce apoptosis of Eca109 cell line partly by up-regulating the expression of p-p38MAPK/Fas/FasL.