| ObjectiveEsophageal carcinoma is a commonly malignant tumor. Search for effective chemotherapy is very important in treating malignant tumors.Norcantharidin is made by changing structrue of Cantharidin which has antineoplasm activity and severe side effects, and has the main active component for antineoplasm. In this study, Ec9706 cells were cultured in vitro and intervented with norcantharidin, then the effects of norcantharidin were studied by morphological observation of cell, analysis of cell cycle distribution in cell apoptosis and the expression of regulatory genes of cell apoptosis. To detect the effect of Norcantharidin on the cell proliferation and apoptosis of Human Esophageal Carcinoma Ec9706 Cell lines, study the expression of Caspase-3,survivin and NF-κB, and to investigate the mechanism of Norcantharidin to induce tumor cell's apoptosis, so as to provide experimental basis for chemotherapy of esophageal carcinoma.Methods 1,After the treatment of norcantharidin on human esophageal carcinoma Ec9706 cell, detect the inhibition of Ec9706 cell growth.Esophageal carcinoma Ec9706 cells were cultured and intervened by norcantharidin with various concentrations(0,5,10,20,40μg/ml) and different times(12h,24h,48h), respectively. Cell growth inhibition was detected by MTT, and acquired the concentration of half inhibition.2,The cell apoptosis and expressions of regulatory genes were detected by FCM.Esophageal carcinoma Ec9706 cells were cultured and intervened by norcantharidin with IC50 concentration and different times(12h,24h,48h), respectively. Cells treated by each concentration and time were collected and fixed in 70% ice-cold ethanol. DNA content and DNA ploidy were detected by FCM. The expressions of regulatory genes were also detected by FCM, then their correlations were analyzed.3,The expression of Survivin,Caspase-3 and NF-κB protein was investigated by the measure of immuno-cytochemistry.Esophageal carcinoma Ec9706 cells were cultured on slides and intervened by norcantharidin with IC50 concentration and different times (12h,24h,48h), respectively. The expressions of regulatory protein of cell apoptosis were observed by immuno-cytochemistry and analysis positive expression rate.Results1 The effects of norcantharidin on cell apoptosis in Ec9706 cells.Norcantharidin inhibited the growth of Ec9706 cells in a dose and time dependent manner. IC50 was gained from the experiment of MTT. The apoptosis cells were also observed on a DNA histogram as subdiploid or pre-G1 peak, pre-G1 peak was especially remarkable at 24h , and the percentage of apoptotic cells increased. The cell cycle distribution changed obviously with treatment of norcantharidin . 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. Finally, a characteristic DNA"ladder"on the agrose gels was observed in norcantharidin-treated cells assayed by agarosegd eletrophoresis.2 The effects of norcantharidin on cell apoptosis and regulatory genes expression in Ec9706 cells.Treated Ec9706 cells with IC50 concentration of norcantharidin for (12,24,48) h, and detected through FCM. Caspase3 protein expressions in treated cells were higher than that in contol cells. Survivin protein expression were lower than that in control cells. NF-κB protein expressions were positive in control cells, the protein expression increased first and then decreased obviously when treated time was adequate. There was positive correlation between Caspase3 and NF-κB (P<0.05), and negative correlation between Caspase3 and Survivin (P<0.05).The expression of regulatory genes of cell apoptosis in Ec9706 cells changed obviously with IC50 norcantharidin treatment for different times(6h,12h,24h)detected by IHC. Caspase3 protein positive expression rate in treated cells were higher than that in contol cells. Survivin protein positive expression rate in treated cells were lower than that in control cells. NF-κB protein expressions were positive in control cells, the protein positive expression rate increased first and then decreased obviously when treated time was adequate.Conclusions1 Norcantharidin inhibited the growth of human Ec9706 cells in a dose and time-dependent manner, norcantharidin induced apoptosis and disturbed cell cycle of Ec9706 cells.2 Down-regulated the expression of Survivin protein , up-regulated the expression of caspase-3, norcantharidin might induce apoptosis of Ec9706 cells through Fas associated caspase pathway and Mitochondrial pathways.3 Esophageal carcinoma Ec9706 cells were inhibited by norcantharidin, this Caspase3 protein expression in treated cells were higher than that in contol cells, while Survivin protein expression were more positive in control cells than in treated cells. NF-κB protein expressions were positive in control cells, the protein expression increased first and then decreased obviously when treated time was adequate. Norcantharidin inhibited the growth of human Ec9706 cells was a process with multiple factor and multiple pathway action, need further research.
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