| ObjectiveEsophageal carcinoma is one of the high incidence in digestive malignant tumor. There are about 300,000 people died in every year. It is popular in China, where the incidence of it is the first in the world. Chemotherapy has become the important method to cure malignant tumors, but the cytotoxicity of many chemical durgs is not specific. At the moment of killing the tumour cell,it un-selective killed normal cell.So to search weak toxicity and side effect antineoplasm activity fortis chemotherapeutics to become hot spot of search. Curcumin is a natumal compound present in turmeric,a rhizome of the plant Curcuma longa lin,it is extensively used as a dietary spice and pigment in cooking. Further studies show that the pharmacological action generally of curcumin,as anti-inflammatory,antioxygen,anticoagulation,to drop blood-fat,anti-arteriosclerosis and anti-tumour,especially curcumin can induce multitude tumour cell apoptosis,to be a kind of anticancer drugs have good development perspective,cause human being paying close attention to it increasingly. There has not been many report about effects of curcumin on esophageal carcinoma cells. In this study, Ec-9706 cells were cultured in vitro and intervented with curcumin , then the effects of curcumin were studied by morphological observation of cell, analysis of cell cycle distribution in cell apoptosis and the expression of regulatory genes of cell apoptosis,so as to provide experimental basis for chemotherapy of esophageal carcinoma.With the establishment of the regulatory molecular mechanism of cell apoptosis, more and more research indicate that tumor is a kind of disease with cell apoptosis. The abnormality of cell apoptosis regulatory genes will result in disturbance of cell apoptosis, lead to excessive proliferation and decreasing apoptosis, then cell growth is out of control at last. In this research, different methods were used to study the expression of cell apoptosis regulatory genes which correlate to each other-inhibitor of apoptosis protein (Livin) , second mitochondria-derived activator of caspase (Smac), and caspase-3. The purpose of this study is to clarify the cascade regulatory mechanism of cell apoptosis in the carcinogenesis of esophageal epithelia.MethodsEc-9706 cells were cultured in the environment of 37℃, 5%CO2 with routine method .The logarithmically growing Ec-9706 cells were used.1 Using light microscope observe the change of morphology: Ec-9706 cells were treated with different concentrations of curcumin,the change of morphology was observed by inverted phase contrast microscope in different time and photographed. 2 The suppressive effects of curcumin on the proliferaition of Ec-9706 cells were evaluated in vitro by MTT colrimetric assay.3 Flow cytometric analysis: Ec-9706 cells were treated with different concentrations of curcumin for 12,24,48 hour. Experimental group and control group cells were harvested and washed twice with PBS. Cells were fixed with ice cold 70% ethanol at 4℃. Precipitates were digested with 0.5% pepsin after centrifugation. And then cells were resuspended in 0.5ml propideium iodide (PI)/RNAase A solution. Cells were incubated in the dark at room temperature for 15 min. The fluorescence emission of stained cells was measured with a flow cytometer. Data were analyzed with Multipcycle software.4 Analysis of caspase-3 proteins by flow cytometry: Ec-9706 cells were treated with different concentrations of curcumin for 12,24,48 hour. At the end of the treatment ,adherent and floating cells were combined and centrifuged, cells were stained by indirect immunofluorescence labing method. A histogram plot of FITC-fluorescence intensity (in logarithmic fluorescence intensity) (χ-axis) versus counts (у-axis) has been shown by flow cytometry. Fluorescence index was used to analysis the expression of caspase-3 proteins.5 The protein expression of regulatory genes of cell apoptosis Caspase3 were observated by immuno-cytochemistry.6 The expression of gene protein (Livin,Smac,caspase-3) were detected by Western blot . Results1 Ec-9706 cells were treated with 0μmol/ml,40μmol /ml,80μmol/ml,120μmol/ml and 160μmol/ml curcumin for12h,24h,48h. Inverted phase contrast microscope observed, comparing with control group cells, the growth of drug-treated group cells were inhibited, cell granulations were increased and thicken,some cells became round and suspension in the medium. All the changes indicated that curcumin induced apoptosis of Ec-9706 cells.2 Ec-9706 cells were treated with 0μmol/ml,40μmol /ml,80μmol/ml,120μmol/ml and 160μmol/ml curcumin for 12h,24h,48h respectively, the growth of cells was inhibited significantly in a time- and dose- dependent fashion. The IC50 of Ec-9706 cells of 12h,24h and 48h were 216.1447μmol /ml,148.0635μmol /ml and 113.8045μmol /ml respectively.(P<0.01). Little influence is found on the growth of cells treated with solvent(P>0.05).3 The analysis of cellular DNA content by FCM showed that there was a sub-G0/G1 peak in the graph of drug-treated groups. That was a typical apoptotic peak,which was not shown in the graph of control groups. The alteration occured in a dose and time dependent manner .After Ec-9706 cells were exposed to curcumin(40,80,120 and 160μmol /ml) for 12h, the apoptosis rates were 6.01±1.04%,9.26±2.11%,13.31±1.54% and 20.16±2.85%respectively , which were significantly higher than control 1.70±0.32% (P<0.05).The apoptosis rates of 24h were 8.74±0.98%,11.63±1.74%,21.67±2.34%,36.17±2.78% respectively, which were significantly higher than control 2.12±0.48% (P<0.01). The apoptosis rates of 48h were 9.14±1.62%,17.74±1.87%,30.82±2.48% and 44.35±3.42% respectively, which were significantly higher than control 2.87±0.54% (P<0.01). The cell cycle distribution changed obviously with treatment of curcumin. The cell number in G0/G1 phase increased gradually, but that in S and G2/M phase decreased gradually. The alteration occured in a dose and time dependent manner.4 Analysis of caspase-3 proteins by flow cytometry: Ec-9706 cells were treated with curcumin (0,40,80,120 and 160μmol /ml) for 12h,24h and 48h. Expressions of caspase-3 protein was increased .Fluorescence index of caspase-3: 12h were(1.00±0.00) (1.05±0.07) (1.18±0.04) (1.23±0.09) (1.72±0.11). 24h were(1.00±0.03) (1.27±0.04) (1.43±0.04) (1.89±0.07) (2.11±0.13). 48h were(1.00±0.02) (1.31±0.05) ( 1.81±0.03) (2.09±0.08) (2.35±0.07). which were significantly higher than control (P<0.01). The alteration occured in a dose and time dependent manner.5 The expression of regulatory genes of cell apoptosis in Ec-9706 cells changed obviously with 40,80,120 and 160μmol /ml curcumin treatment for different times(12h,24h and 48h)detected by ICC. Smac and Caspase-3 protein expression were weak positive in control cells,the protein expression increased obviously in treatment with curcumin.6 The protein expression was detected semi-quantitively by Western blot. Following treating Ec-9706 cells with curcumin for 48h, the protein expression of Smac, Caspase-3 increased gradually, but that of Livin decreased gradually, the alteration occured in a dose dependent manner.Conclusions1 Curcumin can inhibit proliferation and the cell cycle distribution changed obviously , induced apoptosis of Ec-9706 cells.The growth of cells was inhibited and cells apoptosis in a dose and time-dependent manner.2 Ec-9706 cells were a high expression of Livin protein.After treated with curcumin,Livin protein decreased gradually.weaked the effect of negative regulation, induced apoptosis of Ec-9706 cells3 After treated with curcumin,Smac protein expression was a up-reglated,relifed the effect of Livin inhibited Caspase expression ,active the Procaspase-3 to Caspase-3,than induce turmor cells apoptosis.4 The expression of Caspase-3 was increased with the dose and time increasing , active the Procaspase-3 to Caspase-3, split DNA and damage the cells, than induce Ec-9706 cells apoptosis.
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