| Objective:Esophageal carcinoma is a commonly 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. The occurrance of esophageal carcinoma is a process with multiple factor action, polygene change and multiple phase development. The oncogenesis of esophageal squamous epithelia is from normal epithelia, simple hyperplasia, atypical hyperplasia to carcinoma. The atypical hyperplasia is a kind of precancerous lesion, so the key of reducing the incidence and mortality of esophageal carcinoma is monitoring effectively to precancerous lesion and early detection, early diagnosis and early treatment 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 investigation, different methods were employed to study the expression of cell apoptosis regulatory genes which correlate to each other, analyse the DNA ploidy in cell apoptosis. The purpose of this study is to clarify the cascade regulatory mechanism of cell apoptosis in the carcinogenesis of esophageal epithelia.Chemotherapy has become the important method to cure malignant tumors, but the cytotoxicity of many chemical durgs is not specific. Cantharidin is a valuble traditional Chinese medicine which has antineoplasm activity.But it has severe side effects. Norcantharidin is made by changing structrue of Cantharidin, norcantharidin has the main active component for antineoplasm, moreover, it's toxicity to normal cells is very weak. It has lots of effects such as increasing white cell, protecting liver cell and improving immunity, etc. It is used to treat many tumors. There has not been any report about effects of norcantharidin on esophageal carcinoma cells. In this study, Eca-109 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 , so as to provide experimental basis for chemotherapy of esophageal carcinoma.Methods:1 Detection of expression of Fas,Fasl,Fadd,Caspase8 in the carcinogenesis of esophageal epithelia by immunohistochemistry and in situ hybridization.60 cases of esophageal carcinoma and para-carcinoma epithelium were collected after surgical operation and fixed in 4% paraformaldehyde, then proceeded paraffin embedding, routine HE staining and pathological diagnosis. Immunohistochemistry (IHC) was employed to detect the expression of Fas,Fasl,Fadd,Caspase8 protein in 60 cases of esophageal carcinoma, 30 cases of atypical hyperplasia tissues and 30 cases of normal epithelium. In situ hybridization was employed to detect the expression of Fadd,Caspase8mRNA in 60 cases of esophageal carcinoma, 30 cases of atypical hyperplasia tissues and 30 cases of normal epithelium. Furthermore, the correlations between the gene expression and clinicopathological parametes were analysed.2 Quantitive detection of DNA content and expression of cell apoptosis regulatory genes by FCM in the carcinogenesis of esophageal epithelia.30 cases of esophageal carcinoma and 30 cases of para-carcinoma epithelium were collected after surgical operation and fixed in 70% ice-cold ethanol. DNA content and DNA ploidy were detected by FCM. The expression of regulatory genes which correlate with each other closely in cell apoptosis were detected by FCM equipment, then their correlations were analysed.3 The investigation of the effects of norcantharidin on cell apoptosis and regulatory genes expression in Eca-109 cellsThe Eca-109 cellS were cultured, and intervented by norcantharidin with various concentrations and different times(6h, 12h and 24h), respectively. Cell growth inhibition was detected by MTT. The morphological and ultrastructure alterations were observed by light microscope and transmission electron microscope, recpectively.The apoptosis and proliferation index of cells were analysed by FCM. The cell apoptosis was determined by agorase gel electrophoresis. The protein expression of regulatory genes of cell apoptosis including Fas-c-FLIP-Caspase8-Caspase3 were observated by immuno-cytochemistry. The expression of gene protein and mRNA were detected by Western blot and RT-PCR, respectively.Results:1 The expression of Fas,Fasl,Fadd,Caspase8 in esophageal epithelia.From normal esophageal epithelia to atypical hyperplasia tissues and carcinoma tissues, the expression of Fasl protein appeared increasing gradually, the expression of Fas protein appeared decreasing gradually, .The expression of Fas protein in esophageal carcinoma(41/60,68%)was lower than that in normal epithelia(28/30,94%)(P<0.01). The positive rate of Fas protein expression in well differentiated carcinoma was higher significantly than that in moderately and poorly differentiated carcinoma(P<0.01).The expression of Fasl protein in esophageal carcinoma(51/60,85%)was higher than that in normal epithelia(18/30,60%)(P<0.01). The positive rate of Fasl protein expression in well differentiated carcinoma was higher significantly than that in moderately and poorly differentiated carcinoma(P<0.01).The expression of Fadd protein in esophageal carcinoma (38/60,64%) was lower obviously than that in normal epithelia(28/30,96%) (P<0.01). Fadd mRNA expression in esophageal carcinoma(36/60,60%) was lower obviously than that in normal epithelia(27/30,91%), there was no significant diffrence between the positive rate of Fadd protein and mRNA expression in well differentiated carcinoma and that in moderately and poorly differentiated carcinoma( P>0.05).The expression of caspase8 protein in esophageal carcinoma (43/60,71%) was lower obviously than that in normal epithelia(29/30,98%) (P<0.01). Fadd mRNA expression in esophageal carcinoma(40/60,67%) was lower obviously than that in normal epithelia(27/30,92%), The positive rate of caspase8 protein and mRNA expression in well differentiated carcinoma was higher significantly than that in moderately and poorly differentiated carcinoma(P<0.01).In the carcinogenesis of esophageal epithelia, Fas and Fasl,Fadd and Caspase8 gene expression appeared to have positive correlations, but others had not obvious correlation. Except for histological differentiation,Fas,Fasl,Fadd,Caspase8 gene expression were not relevant with other clinicopathological parameters.2 DNA content and expression of cell apoptosis regulatory genes in the lesions of esophageal epithelia.DNA content increased gradually from normal esophageal epithelia, atypical hyperplasia to carcinoma tissue. DNA content in carcinoma (1.35±0.19) was higher significantly than those in normal epithelia (1.00±0.04) and atypical hyperplasia tissue(1.03±0.03 )(P<0.01 or P<0.05). 10 cases of normal epithelium and 20 cases of atypical hyperplasia tissues were all diploid(100%), but 25 cases of carcinoma were heteroploid among 30 cases (84%). There was significant difference of heteroploid rate between esophageal carcinoma and para-carcinoma tissues.There was significant difference between esophageal carcinoma and para-carcinoma tissues of protein expression of Fas,Fasl,Fadd,caspase8,caspase3,c-FLIP gene(P<0.01). There was positive correlation between Fas and Fasl,Fadd and caspase8 gene (P<0.01 or P<0.05), but others did not obviously correlated with other genes(P>0.05).3 The effects of norcantharidin on cell apoptosis and regulatory genes expression in Eca-109 cells.Norcantharidin inhibited growth of Eca-109 cells in a dose and time dependent manner. The IC50 of norcantharidin was 10μg/ml. Treating Eca-109 cells with 10μg/ml norcantharidin for 24h, comparing with control cells, the number of cells decreased obviously, the figure of partial cells changed from polygonal to long fusiform shape with clearer cell outline and small ratio of nucleus to plasm. Distinct apoptosis morphological changes characterized by chromatin condensation, nuclear shrinkage, nuclear cleavage were found in norcantharidin-treated cells by TEM. mitochondria and rough endoplasmic reticulum decreased too, All the changes indicated that norcantharidin induced apoptosis of Eca-109 cells.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 to 20.68±3.12%. 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.The expression of regulatory genes of cell apoptosis in Eca-109 cells changed obviously with 10μg/ml norcantharidin treatment for different times(6h, 12h and 24h)detected by IHC. c-FLIP protein expression were strongly positive in control cells, the protein expression decreased obviously in partial long fusiform cells with 10μg/ml norcantharidin treatment. Fas,Caspase8 and Caspase3 protein expression in treated cells were higher than that in contol cells.The protein expression were detected semi-quantitively by Western blot. Following treating Eca-109 cells with 10μg/ml norcantharidin from 6h, 12h to 24h, the protein expression of Fas, Caspase8 and Caspase3 increased gradually, but that of c-FLIP decreased gradually, the alteration occured in a time dependent manner. Semi-quantitive RT-PCR was employed to detect mRNA expression. Caspase8 and Caspase3 mRNA expression increased obviously. The alteration of mRNA occured in a time dependent manner too.Conclusions:1 The ratio of Fas and Fasl gene changed in esophageal carcinoma, there is significantly positive correlation between Fas and Fasl. Their synergistic action is an important event in oncogenesis of esophageal epithelia, the combinded detection of Fas and Fasl genes probably helps for early prognosis of esophageal carcinoma.2 DNA content increased during the carcinogenesis of esophageal epithelia. The heteroploid rate is one of markers to reflect malignant biological character of esophageal carcinoma.3 We analyzed the expressions of protein and mRNA of Fdaa and caspase8 in squamous carcinogenesis of the esophagus, Fadd and caspase8 may be important roles in death inducing signalling complex. The expressions of protein and mRNA of Fadd and caspase8 decreased gradually, Fadd and caspase8 may play important roles in tumorigenesis of EC.4 We resulted the genes correlated with Fas associated apoptosis signalling pathway from normal squamous epithelia to dysplasin and carcinoma, the ratio of Fas and Fasl gene changed, expressions of Fadd, caspase8 and caspase3 protein decreased gradually, expression of c-FLIP protein increased gradually, as the cascade regulatory machanism of cell apoptosis, Fas - Fasl - Fadd-caspase8- caspase3-c-FLIP plays an important role in the carcinogenesis of esophageal epithelia.5 Norcantharidin inhibited the growth of human Eca-109 cells in a dose and time-dependent manner, norcantharidin induced apoptosis of Eca-109 cells.6 Norcantharidin induced apoptosis of Eca-109 cells through Fas associated caspase pathway, down-regulated the expression of c-FLIP, up-regulated the expression of caspase-3 and caspase8. |
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