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Objective To study the relationship between osteopontin (OPN) gene expression and the development and metastasis of esophageal cancer, and explore the effect of growth inhibitory of artesunate (Art) on regulating the expression of OPN in esophageal carcinoma cells.Methods The expressions of OPN gene and protein were detected in normal esophageal tissue (24 cases), esophageal dysplasia (21 cases) and esophageal squamous cell carcinoma (ESCC) tissue (45 cases) byin situ hybridization and immunohistochemistry, and the relationships between the OPN gene and protein expressions and the pathological features were analyzed with SAS software. After intervention with different concentrations of Art (0, 30, 60, 120mol/L) on ESCC Eca109 cells for 24h, the levels of OPN expression and cell cycle were detected by flow cytometry.Results The expression levels of OPN gene and protein were significantly higher in esophageal cancer tissues than in esophageal dysplasia and normal esophageal tissue (P0.05), but showed a positive correlation with lymph node metastasis and the invasion depth of esophageal cancer (P<0.05). After treatment by Art for 24h, the proliferation index of Eca109 cells and the expression of OPN protein decreased significantly (P<0.01) in a concentration dependent manner.Conclusions The abnormal high expression of OPN gene may participate in the occurrence, invasion and metastasis of esophageal cancer, so can be used as an objective index of metastasis for esophageal cancer. Art inhibits the growth of esophageal carcinoma cells by down-regulating the expression of OPN protein.
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Objective:This study aims to explore the relationship between serum response factor (SRF) expression level and gas-tric cancer progression by detecting SRF expression level in cancer cells. Methods:The SRF gene in SGC-7901 cells was silenced by RNA interference. Transfection efficiency was detected by fluorescence microscopy, cell proliferation by CCK 8 method, SRF gene and protein expression level by real-time polymerase chain reaction and Western blot, and cell cycle by flow cytometry. Results:Cell treat-ment with siRNA-SRF induced significant reduction in SRF mRNA levels. Western blot analysis showed that SRF protein decreased by 40.1%in the siRNA group compared with that in the control group (P<0.05). Compared with the blank, negative, and mock transfection control groups, cell proliferation of the siRNA-SRF group decreased. The inhibition ratio reached 64.24%, as measured by the CCK-8 assay (P<0.05). Treatment with siRNA could block SGC-7901 cell cycle at G0/G1 phase (P<0.05). Conclusion:SRF expression is close-ly associated with gastric carcinoma cell proliferation. SRF protein level detection can provide a certain reference value in evaluating malignant gastric carcinoma progression. SRF is possibly an important target for the prevention and control of gastric cancers.
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Objective To explore the expression of ATP-binding cassette transporter C2(ABCG2) in adriamycin(ADM)-resistant human esophageal cancer cells.Methods The ADM-resistant human esophageal cancer cell(Eca109/ADM) was induced by gradually increasing the ADM concentration in the culture medium of human esophageal cancer cell line(Eca109) and long time screening culture.ABCG2 mRNA and protein of ADM-resistant cells was detected by RT-PCR,flow cytometry(FCM) and Western blot.Drug excretion of Eca109/ADM cells was examined by FCM.The drug resistance index to ADM was detected by MTT.Results The expression of ABCG2 in Eca109/ADM cells was higher than that in Eca109 cells.The drug excretion of Eca109/ADM cells was stronger than Eca109 cells.The Ecal09/ADM cells drug resistance index to ADM was 3.29.Conclusion The ADM-resistant cell line Eca109/ADM was established successfully as an ideal chemoresistant cell model.ABCG2 might be involved in the drug resistance of esophageal cancer cell.
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Objective:To analyze the factors that affect patient prognosis after radical nephrectomy.Meth-ods:A total of 389 cases of renal cell carcinoma treated with radical nephrectomy between January 1 993 and December 2006 were reviewed.All the data were encoded.inserted into an Excel database and then ana-lyzed by SPSS 1 3.0 software.The cumulative survival rates were calculated by life-table method.We as-sessed the impact of multiple covariates on survival time with the Cox Regression model.Results:The patho-logical results showed that 307 cases were clear call carcinoma,51 cases were papillary renal cell carcinoma,21 cases were chromophobic renal cell carcinoma,2 cases were collecting duct carcinoma.and 8 cases were unclassified.One hundred and ninety-eight cases were of T1N0M0, 113 cases were of T2N0M0, 3 cases were of T1N1M0,10 cases were of T2N1M0, 51 cases were of T3N0M0, and 14 cases were of T3N1M0, Two hundred and sixty-eight cases were followed up.The 1-year survival rate was 96.5%,the 3-year survival rate was 90.7%.the 5-year survival rate was 75.7%.and the 10-year survival rate was 65.8%.Multivariable analysis revealed that significant prognostic factors included TNM stage,Robson stage.vena cava and supplementary treat-ment(X2=22.50.P=0.001).The most important prognostic factor was pathological stage(TNM and Robson).The regression coefficients were 0.533 and 0.674,and the relative risk was 1.941 and 2.01 1(P=0.004 and p=0.002).Conclusion:Radical nephrectomy is safe and effective.TNM stage.Robson stage and vena cava are prognostic factors.Supplementary treatment is a protective factor.
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Objective To investigate the effect and possible mechanism of high mobility group box (HMGB) 1 in the development and progress of rheumatoid arthritis.Methods PBMC and serum samples were obtained from 74 RA patients (38 in active stage and 36 in stable stage) and 26 healthy controls.The expression of HMGB1 mRNA and protein was detected by RT-PCR and ELISA.Flow cytometry analysis ( FCM ) was used to detect the expression of Toll-like receptor 4 on PBMC.Results ①The expression of HMGBI mRNA and protein in active RA patients was significantly higher than that in healthy controls and inactive RA patients [2.63 vs 0.71，0.93 and (10.2±1.2) vs (7.5±1.8)，(8.3±1.8) ng/ml，respectively](P＜0.01 ).② The relative expression of TLR4 protein on CD14+ monocytes and CD3+ lymphocytes in active RA patients was increased than that in inactive RA and healthy controls (P＜0.05 or P＜0.01 ).It was also higher in inactive RA than in healthy controls (P＜0.05 or P＜0.01 ).③ Level of HMGB1 protein in serum of RA patients was positively correlated with ESR，CRP，RF，the numbers of tender joints and swollen joints as well as radiographic changes.Conclusion HMGB1 can be synthesized and released by PBMC of active RA patients，and then bind to TLR4 of PBMC to promote inflammatory responses and bone erosion.
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Objective To investigate the effect and possible mechanisms of high mobility group box (HMGB) 1 on the proliferation of RSC-364 synoviocytes. Methods ① RSC-364 cells stimulated by 10 μg/L TNF-α and cells of the normal control groups were collected at 6, 12, 24 h respectively in vitro. HMGB1mRNA and protein was detected by reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry (ICC); ②RSC-364 cells induced by 10 μg/L HMGB1 were collected in 6, 12, 24 h respectively, so did normal control group cells in vitro. The expression of signal transducer and activator of transcription (STAT) mRNA 1 was detected by RT-PCR. The expression of STATland SOCSI proteins were detected by ICC and flow cytometry analysis (FCM). The expression of PCNA was detected by ICC. Results ① Compared with the control group, TNF-α markedly up-regulated HMGBI mRNA at 6, 12, 24 h respectively [0.86, 0.92, 1.06 vs 0.70, P<0.01 ], as well as protein expression level. Positive signal of HMGB1 proteins was not only expressed in nuclear but also in cytoplasm after stimulation. ② Compared with normal group, HMGBI increased the expression of P-STAT1 mRNA and protein at 6, 12, 24 h respectively [0.30, 0.69, 1.05 vs 0.24, P<0.01 ] and [1.34±0.09，1.55±0.16，1.74±0.13 vs 1.00±0.15，P<0.01]. The expression of SOCSI protein increased significantly in HMGB1 group at 6 and 12 hours ( 1.43±0.10 vs 1.58±0.05), but it decreased at 24 hours (1.24±0.15). ③The expression of p-STATI protein was negatively correlated with that of SOCS1 protein. Conclusion HMGB1 appears to be an important mediator in the proliferation of RSC-364 cells, partly by up-regulating the expression and aetivity of p-STAT1.
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Objective To investigate the effect and possible mechanism of arsenic trioxid(As2O3)on proliferation of RSC-364 synoviocyte lines stimulated with TNF-?.Methods RSC-364 synoviocytes were cultured with standard medium as control group or medium supplemented with 10 ?g/LTNF-? and different concentrations of As2O3 respectively. MTT assay were carried out to study cell proliferation. Proliferation index (PI) and cell cycle were detected by flow cytometry (FCM). RT-PCR was used to detect the mRNA expression of High mobility group box chromosomal protein (HMGB)1. HMGB-1 and proliferation cell nuclear antigen (PCNA) proteins were detected by immunocytochemistry and FCM. Results (1)As2O3 inhibited proliferation of cell lines stimulated by TNF-? time-dependently and dose-dependently. (2)Compared with normal group, TNF-? up-regulated HMGB-1 protein and mRNA as well as PCNA protein. HMGB-1 protein was not only in nuclear but also in cytoplasm by immunocy-tochemistry. As2O3 down-regulated mRNA and protein of HMGB-1 in a dose-dependent manner; so did PCNA proteins (P
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Objective To investigate the effects of the gene expressions of FAS、FASL、FADD and caspase-8 in esophageal carsinogenesis. Methods Immunohistochemical method was applied to detect the expression of FAS、FASL、FADD and caspase-8 proteins in esophageal epithelium. In situ hybridization method was applied to detect the expression of FADD and caspase-8 mRNA in esophageal epithelium. Results The positive rates of FAS, FADD and caspase-8 proteins and FADD、caspase-8 mRNA were decreased from normal epithelium to dysplasia and carcinoma tissues gradually. The positive rates of FASL protein were increased from normal epithelium to dysplasia and carcinoma tissues gradually. There was very significant statistical difference between carcinoma and normal epithelium(P
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Objective To investigate the effects of selective COX-2 inhibitor nimesulide on growth inhibition,apoptosis and expression of COX-2 of human esophageal carcinoma Eca-109 cell line; and analyzed the correlation with the anti-oncogene,P277~(kip1). Methods MTT assay was used to detect the proliferation of Eca-109 cell. Apoptosis and cell cycle were determined by electronic microscopy and flow cytometry. The expression of COX-2 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR),the protein expression of COX-2 and P277~(kip1) were examined by Western blot analysis. Results Nimesulide significantly inhibited the proliferation of Eca-109 cell line in a time-and dose-depenent fashion; increased the proportion of cells in the G_0/G_1 phase and induced apoptosis of the cells in a dose-dependent(manner). Meanwhile,nimesulide can down-regulated the expression of COX-2 and up-regulated the expression of P277~(kip1) protein.Conclusion Nimesulide can inhibit the proliferation of Eca-109 cells,cause G_0/G_1 phase cell cycle arrest and induce apoptosis.The mechanism is probably explained with down-regulation of the expression of COX-2 and up-regulation of P277~(kip1) expression.
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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
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Objective To investigate the expressions of EphA2 and its ligand EphrinA1 and their relationship with angiogenesis in gastric carcinoma. Methods Expressions of EphA2,EphrinA1 and CD34-stained microvessel density (MVD) were detected by immunohistochemical assay in gastric carcinoma tissues,adjacent tissues (1.5 to 2 cm from the mass) and normal gastric mucosa (5 to 10 cm from the mass) from 82 cases. The correlations among EphA2 and EphrinA1 expressions,MVD and clinic pathological features were analyzed. Results EphA2,EphrinA1 expressions and MVD in gastric carcinomas tissues were significantly higher than those in adjacent tissues and normal gastric tissues (P
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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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<p><b>OBJECTIVE</b>To explore the molecular events and mechanism in the carcinogenesis of esophageal epithelium in the high incidence area of esophageal carcinoma.</p><p><b>METHODS</b>Epithelial cells collected from the high incidence area of esophageal carcinoma were used to detect DNA content and ploidy by propidium iodide(PI) stain. The expressions of p53, p16 and cyclin D1 were stained by indirect immunofluorescence of fluorescein isothiocyanate(FTTC), which were detected by flow cytometry (FCM).</p><p><b>RESULTS</b>During the process of carcinogenesis, DNA content increased significantly. The diploid cells decreased while heteroploid cells increased sharply, with a heteroploidy rate of 84.2%. At the same time, the p53 protein accumulated and p16 was deleted. The positive rates of p53 and oncogene cyclin D1 were both 100%(5/5, 6/6) in the cancer group.</p><p><b>CONCLUSION</b>In the early carcinogenesis of esophageal epithelium, DNA content and heteroploidy rates increase with tumor suppressor gene p16 deletion and p53 protein accumulation while oncogene cyclin D1 is overexpressed. Multiple molecular events have already occurred when esophageal carcinoma develops.</p>
Subject(s)
Humans , Cyclin D1 , Metabolism , Cyclin-Dependent Kinase Inhibitor p16 , Metabolism , DNA, Neoplasm , Metabolism , Esophageal Neoplasms , Genetics , Metabolism , Pathology , Flow Cytometry , Gene Expression , Precancerous Conditions , Genetics , Tumor Suppressor Protein p53 , MetabolismABSTRACT
Cell DNA content in heart, liver, kidney of rats were analysed by flow cytometer at different postmortem interved. The rsults show that mean cellular DNA content is 99.5% at 6 hours, 91.3% at 12 hours, 87.1% at 18 hours, 81.3% at 24 hours, 76.7% at 30 hours, 74.3% at 36 hours, 72.3% 48 hours as compared with that at o hours The results ingicates that the quantitative determination of cell DNA in the tissues, mentioned above may provide an objective and reliable approach for the estimation of postmortem time.
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Aim To investigate the effects and possible mechanism of valdecoxib on apoptosis of cancer in nude mice.Methods The tumor model was established by inoculating 2?106 cell both in the left and right armpit respectively.The mice were divided randomly into control group and valdecoxib group(20 mg?kg-1?day-1).Valdecoxib was dissolved in carboxymethylcellulose sodium and administered from the second day after inoculation.The mice were killed after 4 weeks.The volumn and inhibitory rate were calculated according to the length and width of xenograft tumor.H.E staining was used to observe the cell structure of the stomach and colon.The apoptotic rate was detected by FCM.The expression of COX-2,c-jun and c-fos protein was detected by FCM and immunohistochemical staining.Total RNA was extracted with Trizol method and the expression of COX-2 mRNA was detected by RT-PCR.Results(1) The body weight of nude mice was higher in valdecoxib treated group in a time-dependent manner.(2) Valdecoxib inhibited the growth of tumor.The weight of tumor was decreased from(1.43?0.52)g in control group to(0.93?0.53)g in valdecoxib treated group.The ratio of inhibition on the growth of tumor was 45.80%.(3) Valdecoxib increased the apoptosis rate from(14.15?0.48)% in control group to(29.80?6.35)% in treated group.(4) The expression of COX-2 mRNA and protein decreased in treated group compared with that in control group.FCM and immunohistochemical staining showed that the expressions of c-jun and c-fos were increased in valdecoxib treated group.There was statistical significance compared with control group.(5) There was significantly negative correlation between the ratio of apoptosis and the expression of COX-2(r=-0.726,P=0.008);there was significantly positive correlation between the ratio of apoptosis and the expressions of c-jun and c-fos protein respectively(r=0.603,0.813;P=0.038,0.001);(6) Valdecoxib did not affect cell structure of stomach and colon.Conclusions valdecoxib inhibits the growth of the xenograft tumor in nude mice and induces the apoptosis.Decreasing expression of COX-2 and up-regulating the expressions of c-jun and c-fos may be one of the mechanisms for the apoptosis.
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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 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.
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Objective:To investigate specific cellular immune response to the HPV16E7 prophylactic vaccine in mice.Methods:BALB/c mice were randomly divided into 3 groups:experimental group (treated with pcDNA3.1-HPV16E7),control group Ⅰ(treated with pcDNA3.1) and control group Ⅱ(treated with N.S.).Mice were injected (i.m.) pcDNA3.1-HPV16E7,pcDNA3.1 and N.S. one time per week,respectively.After three immunization,the blood samples from eye sockets and the supernatant cultured of spleen cells were taken for measurement IFN-? and the number of CD4 +?CD8 +T-lymphocyte by ELISA and FACS assay.Antigen-specific splenocyte proliferation assay in vitro was detected by MTT method.Results:The splenocytes actively proliferated,the number of CD4 +T lymphocyte and the quantitation of IFN-? in spleen and serum in the experimental group were significantly higher than the control group Ⅰ and Ⅱ.Conclusion:The pcDNA3.1-HPV16E7 DNA vaccine can induce specific cellular immune response in BALB/c mice.
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Nuclear DNA content in 103 cases of malignant and benign breast tumours were determined by means of flow cytometry. It was found that DNA in all the benign lesions showed diploids. In contrast, only 32.3 percent of the cancers had diploids, while 62.3% had additional DNA aneuploids including near-diploids (18.4%); triploids (22.3%); and tetraploids (8.2%) etc. Both the cellular proportion in S-phase and DNA index were significantly higher in malignant breast tumours than those in benign ones (P0.05). The results indicate that the analysis of DNA content is a useful and objective adjunct in the assessment of proliferative activity and biological behavior on breast tumour.