| Objective Esophageal carcinoma is a common malignant tumor throughout the world. There are about 300,000 people die of it every year. It is popular in China, where the incidence of it is the first in the world. The oncogenesis of esophageal epithelia is a process with multiple factor action, polygene change and multiple phase development. In the process, esophageal squamous epithelium changes from normal epithelium to simple hyperplasia, atypical hyperplasia and carcinoma in turn. The atypical hyperplasia is precancerous lesion. The key point of reducing the incidence and mortality of esophageal carcinoma is monitoring effectively to precancerous lesion and early detection, early diagnosis and early treatment to esophageal carcinoma. After establishing of the molecular mechanism of cell cycle regulation, more research indicates that tumor is a kind of disease with cell cycle abnormality. The abnormality of cell cycle regulatory genes results in disturbance of cell cycle, leads to excessive cell proliferation but decreasing cell apoptosis, cell growth is out of control at last. In the present study, a variety of methods were used for estimation the expression of cell cycle regulatory genes which correlate closely each other, determination the DNA ploidy and cell phasic distribution in cell cycle, for the purpose of clarificating the cascade regulatory mechanism of cell cycle, cyclinE-CDK2-p21WAF1-Rb-E2F, in the oncogenesis of esophageal epithelia. Chemotherapy has become the important method to cure malignant tumor, but many chemical durgs have not enough specific cytotoxicity. Ginseng is a valuble traditional Chinese medicine, which has lots of biological activities such as antineoplasm, antisenility and antiradiation and so on. Ginsenoside is the main active component in ginseng for antineoplasm. Ginsenoside-Rh2 (GS-Rh2) has the strongest inhibition to cell proliferation among all kinds of ginsenosides. It can inhibit tumor cells growth by inducing cell differentiation and apoptosis, moreover, it's toxicity to normal cells is very weak. Up to now, there has not been any report about effects of GS-Rh2 on esophageal carcinoma cells. In this study, Eca-109 cells were cultured in vitro and intervented with GS-Rh2, then the effects of GS-Rh2 were studied by morphological observation of cell, analysis of cell phasic distribution in cell cycle and detection of the regulatory genes expression of cell cycle, so as to provide experimental basis for chemotherapy of esophageal carcinoma. Methods 1. Detection of expression of cyclinE, CDK2 and p21WAF1 gene in the lesions of esophageal epithelial by immunohistochemistry and in situ hybridization Esophageal carcinoma tissues and para-carcinoma tissues (atypical hyperplasia and normal epithelium) were obtained from 48 patients with primary esophageal carcinoma who underwent resection of esophageal SCC. All samples were fixed in 4% paraformaldehyde overnight, dehydrated in graded ethanol, and embedded in paraffin, performed routine HE staining and pathological diagnosis in turn. The expression of cyclinE, CDK2 and p21WAF1 protein and mRNA was determined by immunohistochemistry and in situ hybridization respectively. Furthermore, the relationships among the three genes each other and the correlations with clinicopathological parametes were analysed. 2. Quantitive detection of DNA content and regulatory genes expression of cell cycle by FCM in the oncogenesis of esophageal epithelia 30 cases of esophageal carcinoma and para-carcinoma tissue were obtained after surgical operation of esophageal SCC and fixed in 70% ice-cold ethanol. DNA content and the distribution of the cells in various phases of the cell cycle were detected quantitatively by propidium iodide (PI) staining, DNA ploidy and cell proliferation index (PI) were analysed. Theexpression of regulatory genes, cyclinE, CDK2, p21WAF1, p53, Rb and E2F, which correlated closely each other in cell cycle, were detected by indirect immunofluorescence of fluorescein isothiocyanate (FITC) using flow cytometry (FCM) equipment, then the relationships among them were analysed. 3. Detection of regulatory gene expression of cell cycle in esophageal carcinoma by RT-PCR and western blot Fresh samples were obtained from 22 patients who underwent resection of esophageal SCC. All samples were put in liguid nitrogon immediately, which consisted of esophageal carcinoma tissues, para-carcinoma tissues and normal epithelia at cutting edge. The whole cell protein was extracted, then protein quantification and western blot were performed for detection of the protein expression of regulatory genes such as cyclinE, CDK2, p21WAF1, Rb and E2F, β-actin was the internal standard in the procedure. Total RNA were extracted, then the mRNA expression of regulatory genes were detected by semi-quantitative RT-PCR. GAPDH, a house-keeping gene, was the internal standard. All results were analysed using LabWorks 4.5 analysis software. 4. The investigation of the effects of ginsenoside Rh2 on cell cycle and regulatory genes expression in Eca-109 cells The Eca-109 cell were cultured in vitrol routinely, and intervented with ginsenoside-Rh2 under various concentration (5, 10, 20, 40μg/ml) and different time (24, 48 and 72h) respectively. Cell growth inhibition was detected by MTT. The morphological and ultrastructure alterations were observated using light microscope and transmission electron microscope recpectively. The phasic distribution and proliferation index of cell in cell cycle were analysed by flow cytometry. The protein expression of regulatory genes of cell cycle including cyclinE, CDK2, p21WAF1, Rb and E2F were observated by immunocytochemistry. The expression of gene protein and mRNA were detected semi-quantitively by western blot and RT-PCR respectively. Furthermore, CDK2 kinase activity was assayed by immunoprecipitation, and the relationship between CDK2 kinase activity andgene expression were analysed. Results 1. The expression of cyclinE, CDK2 and p21WAF1 gene in the lesions of esophageal epithelia From normal esophageal epithelia to atypical hyperplasia tissues and carcinoma tissues, the expression level of cyclinE and CDK2 gene displayed rising tendency. The level of cyclinE protein was markedly increased in esophageal carcinoma tissues(26/48,54.2%), as compared with normal epithelia (1/17,5.9%) (P<0.01) and atypical hyperplasia tissues(9/31,29.0%) (P<0.05). In particular, the level of cyclinE protein in well differentiated carcinoma tissues was significantly higher than in the other histological types such as moderately and poorly carcinoma tissues, various grades of atypical hyperplasia tissues and normal epithelia. cyclinE mRNA expression was significantly different between eophageal carcinoma tissues (29/48,60.4%) and normal epithelia (5/17, 29.4%) (P<0.05). Further analying, cyclinE mRNA in well differentiated carcinoma (13/20,65.0%)was elevated, the differences between it and both of normal epithelia (5/17,29.4%) and mild atypical hyperplasia tissues(6/19, 31.6%) were statistically significant (P<0.05). Similarly, cyclinE mRNA expression level was significantly higher in well differentiated carcinoma than in moderately(6/10,60.0%)and poorly one(10/18,55.6%), but the differences were statisticlly not significant (P>0.05). In addition to, the expression level of cyclinE mRNA in severe atypical hyperplasia tissues(5/6,83.3%)was higher than in normal epithelia and mild atypical hyperplasia respectively (P<0.05). CDK2 protein was found at high levels in esophageal carcinoma tissues(33/48,68.7%), as compared with normal epithelia(0/17,0%) and atypical hyperplasia tissues(5/31,26.1%)(P<0.01). Similarly, expression level of CDK2 mRNA was incresed in esophageal carcinoma tissues(22/48,45.8%), the difference between it and atypical hyperplasia tissues (7/31,22.6%)was significant(P<0.05), the difference was particularly significant between mild atypical hyperplasia(2/19,10.6%) and poorly differentiatedcarcinoma (9/18,50.0%) (P<0.01). p21WAF1 protein overexpression was frequently found in esophageal carcinoma (44/48, 91.7%), atypical hyperplasia tissues(29/31,93.6%) and normal epithelia(15/17,88.2%), the differences among them were statistically not significant (P>0.05). p21WAF1 mRNA expression was detected in 26 of esophageal carcinomas (26/48, 54.2%), the difference was not significant between it and both of atypical hyperplasia tissues (21/31,67.7%) and normal epithelia (9/17,52.9%)(P>0.05). The mRNA level of cyclinE, CDK2 and p21WAF1 displayed strong staining intensity in multi-nucleus tumor giant cells. In the oncogenesis of esophageal epithelia, cyclinE and CDK2 gene expression exhibited a significant positive correlation, but there was no obvious relationship between expression of p21WAF1 and cyclinE or CDK2. Except histological differentiation, the expression of cyclinE, CDK2 and p21WAF1 gene was not correlated with other clinicopathological parametes. 2. DNA content and expression of cell cycle regulatory genes in the lesions of esophageal epithelia DNA content was increased gradually from normal esophageal epithelia, atypical hyperplasia to carcinoma (F=6.404,P<0.01). Which in carcinoma (1.15±0.19) was elevated significantly, as compared with normal epithelia (1.00±0.04)and atypical hyperplasia tissues(1.03±0.03 )(P<0.01 or P<0.05). 6 normal epithelia and 24 atypical hyperplasia tissues were all diploid(100%), heteroploid was detected in 9 of the carcinomas (3/30, 30%). Difference in heteroploid rate between esophageal carcinoma and para-carcinoma tissue was statistically significant (χ2=10.588,P<0.01). The cell percentage of G0/G1 phase was lower in carcinoma (70.79±10.18) than in para-carcinoma tissue(78.03±6.59)(P<0.01), in contrast, both S phase (18.03±7.25)and G2/M phase (11.18 ±4.43) in carcinoma tissues were higher than in para-carcinoma tissue respectively(14.09±4.10 and 7.89±3.27 repectively) (P<0.01 and P<0.05), proliferation index was markedly increased in carcinoma(29.21±10.18) comparing with para-carcinoma tissue(21.97±6.59)( P<0.01).Differences in protein expression levels of cyclinE, CDK2, p53 and E2F gene between esophageal carcinoma and para-carcinoma tissue were significant (P<0.01). But the expression levels of p21WAF1and Rb protein were not statistically different between carcinoma and para-carcinoma tissues (P>0.05). There were positive correlations among cyclinE, CDK2,p53 and E2F gene each other(P<0.01 or P<0.05), but they were not obviously correlated with p21WAF1 and Rb gene(P>0.05). 3. The expression of cell cycle regulatory genes in esophageal carcinoma by RT-PCR and western blot From normal esophageal epithelia at cutting edge to para-carcinoma and carcinoma tissue, the protein expression level of cyclinE, CDK2, p21WAF1, and E2F showed increasing tendency. Differences in cyclinE or CDK2 protein expression among the three groups were statistically significant (P<0.01). p21WAF1 and E2F protein expression was elevated in carcinoma, but the difference between normal epithelia at cutting edge and para-carcinoma tissue was not significant(P>0.05). Rb protein expression was decreased gradually from normal esophageal epithelia at cutting edge to para-carcinoma and carcinoma tissue, but the difference among the three groups was not significant (P>0.05). The protein expression of cyclinE, CDK2, p21WAF1, and E2F gene displayed positive correlations each other (P<0.01 or P<0.05). The levels of mRNA expression were proportional to protein expression. From normal esophageal epithelia at cutting edge to para-carcinoma and carcinoma tissue, the mRNA expression levels of cyclinE, CDK2, p21WAF1, and E2F showed rising tendency, which in carcinoma tissues was higher obviously than in both para-carcinoma tissues and normal epithelia at cutting edge (P<0.01). The mRNA levels of cyclinE and E2F were higher a little in para-carcinoma tissues than in normal epithelia at cutting edge (P<0.05), but differences in CDK2 and p21WAF1 between the two groups were statistically not significant (P>0.05). Rb mRNA expression showed decreasing tendency from normal esophageal epithelia at cutting edge to para-carcinoma and carcinoma tissue, which in normal epithelia at cutting edge was markedlyhigher than in the two other groups (P<0.01). Statistically analysis clearly showed that there were positive correlations among mRNA expression of cyclinE, CDK2, p21WAF1 and E2F gene each other, but they were displayed inverse correlations with Rb gene respectively. 4. The effects of ginsenoside Rh2 on cell cycle and regulatory genes expression in Eca-109 cells Ginsenoside Rh2 inhibited Eca-109 cells growth in dose and time dependent manner. The IC50 of GS-Rh2 was 20μg/ml. Treating Eca-109 cells with 20μg/ml GS-Rh2 for 72 hours, as compared with control cells, the number of cells and nucleus division phase were 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. Observing the ultrastructure, the nucleus diminished and cytoplasm augmented, heterochromatin increased, the number of mitochondria and rough endoplasmic reticulum increased too, some of rough endoplasmic reticulum were dilated so much that they formed pool. All the changes indicated that GS-Rh2 induced differentiation of Eca-109 cells. Furthermore, the cell phasic distribution in cell cycle changed obviously with treatment of GS-Rh2. The cell number in G0/G1 phase was increased, but which in S and G2/M phase was decreased gradually. The alteration was in dose and time dependent manner. The expression of regulatory genes of cell cycle in Eca-109 cells changed obviously with 20μg/ml GS-Rh2 treatment for different times(24, 48 and 72hours) by immunocytochemistry detection. cyclinE and CDK2 protein showed stronger positive staining in control cells, the protein expression was decreased obviously in partial long fusiform cells with 20μg/ml GS-Rh2 treatment. E2F protein expression in treating cells was weaker than in contol cells, on the contrary, p21WAF1and Rb protein level were elevated in treating cells than in control cells. The protein expression was detected semi-quantitively by western blot. After treating Eca-109 cells with 20μg/ml GS-Rh2 from 24h, 48h to 72h, the protein levels of cyclinE, CDK2 and E2F were decreased gradually, but whichin p21WAF1 and Rb were increased gradually, the alteration was in time dependent manner. Semi-quantitive RT-PCR was employed to detect mRNA expression. cyclinE and CDK2 mRNA expression were decreased obviously after treating for 24h, p21WAF1 mRNA expression was increased slightly, but Rb and E2F were not significantly changed. After treating for 48 hours, Rb mRNA expression was increased markedly, whereas E2F mRNA expression was decreased obviously. The alteration was in time dependent manner too. Moreover, with the treatment of 20μg/ml GS-Rh2, the CDK2 kinase activity of Eca-109 cells was reduced gradually from 24, 48 to 72 hours, which was in time dependent manner, the difference between treating cells and control cells was significant (P<0.01). Furthermore, statistical analysis showed that CDK2 activity was highly correlated with CDK2 protein and mRNA expression. (r=0.702 and 0.767 respectively,P<0.01). Conclusions 1. The abnormal expression of cyclinE gene is an early event during the oncogenesis of esophageal epithelia. It probably becomes one of the diagnosis markers of well differentiated esophageal carcinoma. cyclinE and CDK2 gene overexpression are frequently found in esophageal carcinoma, there is significant positive correlation between them. Their synergistic action is an crucial event in esophageal carcinoma development, the combinded detection of cyclinE and CDK2 genes provides new insight for early prognosis of esophageal carcinoma. 2. DNA content displays rising tendency during the oncogenesis of esophageal epithelia. The heteroploid rate is a potent marker that reflects malignant biological character of esophageal carcinoma. The cell phasic distribution in cell cycle changes markedly in esophageal carcinoma, the cell percentage during S phase and G2/M phase are increased obviously. Cell proliferation index is elevated in carcinoma. 3. It is confirmed at different levels of gene expression and by a variety of methods that the cascade regulatory machanism of cell cycle, cyclinE-CDK2 -p21WAF1-Rb-E2F, plays an crucial action in the oncogenesis of...
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