| Esophageal carcinoma is a kind of most common malignant tumor in China with high degree of malignity. Despite the persistent development of treatment methods in recent years, the long-term follow-up result of esophageal carcinoma, on a whole, is still unsatisfied and the prognosis is very inclement with high morbility and mortality (overall 5-year survival rates less than 10%). So preventive measure may be the best way to reduce the incidence of esophageal carcinoma in current. Epidemiological data showed that early recognition, diagnosis and treatment of cancer or preneoplastic lesions is the main measure to reduce the mortality of esophageal cancer, and seeking the drug which can reverse the development of prenoplastic lesions appears to be a useful method. In recent years, the effect of traditional Chinese medicine on cancer prevention has aroused more and more intreasts and many studies have proved that some Chinese herbs may prevent esophageal cancer. Triterpenes compound of Cortex Periplocae (TCCP) is an active component of Cortex Periplocae with anti-tumor and immunoregulation activity which is developed by our laboratory. TCCP has the significant inhibitory effect on tumor proliferation such as stomach cancer, hepatocarcinoma, or breast cancer as well as esophageal carcinoma cells in vitro. However, a further research need to be developed for whether it can reverse the development of prenoplastic lesion of esophagous in vivo.In this present study, we treated F344 rats with the esophageal carcinogen, N-nitrosomethylbenzylamine (NMBA), thrice per week for 5 weeks to establish a model of human esophageal squamous cell carcinoma. Histochemistry, immunohistochemistry, RT-PCR and western blot technique were used in this study to investigate the variable trends of expression and correlation of some proteins and genes interrelated with abnormal proliferation of esophageal epithelium cell during periods of esophageal tumorigenesis induced by NMBA and the effect of TCCP. The purpose of the study is to screen out an applicable and effective esophageal carcinoma preventive agent of traditional Chinese medicine, to offer academic foundation for further clinic study and to spread out its application.Part 1 The observation of tumor-inhibitory effect of TCCP on the nude mice bearing esophageal carcinoma cellsObjective: To observe the inhibitory effect of TCCP on the nude mice bearing esophageal squmous carcinoma cells and explore the inhibitory mechanism in vivo.Methods: Eca109-luc stablely expressing luciferase were inoculated subcutaneously into the hind flank region of nude mice to establish tumor model. Nude mice were randomly grouped until tuomr's length reached 5-7 mm. One group of mice (n=7) was treated i.s. with NS. A second group of mice (n=7) was treated i.s with TCCP (20mg/kg) thrice per week. The course of all the treatments would last for 4 weeks. Tumor growth were monitored with in vivo imaging system. Cell apoptosis in vivo were analyzed by flow cytometry. Western blot was used to investigate protein expression of Survivin.Results: 1 Compared with NS control group, the tumor growth of the nude mice bearing Eca109-luc was significantly inhibited by TCCP treatment (1.38±0.36 g vs 0.82±0.14 g), with the inhibitory ratio 40.7%.2 There was no significant difference in photon number detected by in vivo bioluminescence imaging between NS control group and TCCP group before treatment with TCCP (5.02E+06±1.03E+06, 5.47E+06±1.12E+06 respectively)( P>0.05). After treatment with TCCP, the average photon number of the TCCP group were decreased by 7.65E+06±2.14E+06,6.18E+07±1.36E+07,1.70E+08±1.14+08 and 5.53E+08±1.01E+08 at weeks 1, 2, 3 and 4, respectively, significantly lower than NS control group (6.48E+07±1.25E+07,3.65E+08±1.42E+08,7.64E+08±3.02E+07 and 3.34E+9±4.78E+08, respectively) (P<0.01).3 FCM assay for the apoptosis rate of tumor cells in vivo showed that there was a significant difference between the NS control group (11.6±1.0%) and the TCCP group (32.5±1.2%) (P<0.05).4 Gene expression of Fas in TCCP treated mice was significantly higher than that of NS group(P<0.01), while survivin mRNA was significantly decreased.5 It was observed that the protein expreesion of Fas in the tumor tissues of TCCP group was significantly higher (0.725±0.046), as compared with that of NS control group (0.297±0.026)(P<0.01), while the protein expreesion of Survivin was significantly decreased (0.052±0.003), as compared with that of NS control group (0.387±0.016)(P<0.05).Conclusions: Cell growth in vivo was inhibited significantly by TCCP which might exert its anti-tumor activity through Fas/FasL pathway related to reduction of survivin expression, and then inducing apoptosis of tumor cells.Part 2 Effect of TCCP on NMBA-induced rats esophageal carcinogenesis and its mechanism1 The establish of rat model of esophageal preneoplastic lesion and the effect of TCCP on the cacinogenesisObjective: To investigate the effect of TCCP on esophageal pathology , proliferating cell muclear antigen (PCNA) expression and cell cycle regulatory components in NMBA-induced rat tumorigenesis.Methods: 165 male F344 rats (5-6 weeks of age) were randomly divided into five experimental groups according to the different regiments. It consisted of the following groups: (1) normal control groups (group 1, n=15): rats were raised rountinly; (2) soya oil control groups (group 2, n=15): rats were injected with soya oil 1ml/kg intramuscularily (i.m.); (3) NMBA control groups (group 3, n= 45): rats were injected with NMBA 0.5mg/kg only; (4) Low dose TCCP treatment groups (group 4, n=45): rats received NMBA 0.5mg/kg s.c. plus TCCP 20mg/kg i.m.; (5) High dose TCCP treatment groups (group 5, n=45): rats received NMBA 0.5mg/kg s.c. plus TCCP 10mg/kg i.m..The administration of drugs was scheduled as below: thrice per week for 5 weeks. At 9, 15 and 25 week, 5 rats from groups 1 and 2 and 15 rats from groups 3, 4 and 5 were euthanized by pentobarbital sodium and subjected to gross necropsy. The esophagus of each rat was excised and opened longitudinally. Then esophagus were fixed in 10% phosphate-buffer formalin solution, and routinely embedded in paraffin for HE staining to observe the pathological changes of esophageal tissues, while the expression of PCNA, cyclinD1 and CDK4 was measured by immunohistochemistry.Results: 1 General observations. The mean body weights and food consumption in all rats were not significantly different throughout the bioassay. At the end of the bioassay rats treated with NMBA were found in low spirits with shaggy fur. However, body weights still increased.2 General observations of esophagi of rats. There were no abnormal changes in normal and soya oil group among bioassay. Changes were observed in NMBA-induced rats as following: (1) esophageal mucosa becoming incrassation with color white and scabrities; (2) white patch. there were some pathologic white patch changes on the surface of esophageal mucosa just like dot, circle, orbicular-ovate or strip with size about 0.1-0.2 mm and the area of the pathologic changes increased at the end of bioassay. (3) papilloma. At week 9 of the bioassay, esophagi had pathologic changes like incrassation and white patch. At weeks 15 and 25 of the bioassay, 20% to 60% of the esophagi, respectively, had papillomas. Mean number of papillomas per rat is 0.93 at week 25.TCCP showed significant inhibitory effects on the incidence of the papilloma in group 4 to 20% ( P<0.01), and the mean number of papillomas was also decreased to 0.27. In group 5, the decrease of incidence and number of the papilloma was not statistically significant (33.3%, 0.33, respectively). There was also no statistically significant between group 4 and group 5.3 The results of histopathologic examination of esophagi. The preneoplatic lesion of esophageal tissues was evaluated. At weeks 9, 15 and 25, the incidence of preneoplastic lesion in groups 3 was 20.0%, 46.7% and 93.3 %, respectively. TCCP markedly inhibited rat esophageal preneoplastic lesion, the inhibitory rates at 25 weeks were 64.3% in group 4 and 50% in group 5, respectively (P<0.05).4 There were a markedly increase in the expression of PCNA in NMBA control group (week 9: 213.17±29.74; week 15: 268.35±39.56; week 25: 327.24±28.19), as compared with that in normal control group(167.96±20.16,170.76±14.79,172.49±17.49, respectively)( P <0.05). TCCP significantly decreased the expression of PCNA in groups 4 (185.28±22.98,200.56±28.19,282.23±37.43) and group 5 (189.27±22.10,209.73±28.88,305.60±17.46) (P<0.05).5 The expreesion of CDK4 and cyclin D1 were not found in normal mucosa of esophagi of rats in normal control and soya oil control group, but were induced by administering NMBA. Positive expression of cyclinD1 and CDK4 increased gradually and reached the peak at 25wk. There were significant positive correlations between the two protein expression (r=0.9631, P<0.05). Treatment with TCCP could significantly decrease the protein expression of CDK4 at weeks 9, 15 and 25 (P<0.05). And there was significant difference between the high TCCP group and low TCCP group at weeks 15 and 25. TCCP could act a similar effect on the expreesion of cyclinD1.Conclusions: NMBA (0.5mg/kg) can sucessfully induced F344 rats model of esophageal preneoplastic lesion. Typical histophathologic changes was examined in the NMBA-treated rats. TCCP may restrain the high expression of PCNA, while alleviate the esophageal preneoplastic lesion of rats induced by NMBA. TCCP can also inhibit the uncontrolled proliferation of esophageal epithelia cells and regulating cell-cycle process.2 The effect of TCCP on wnt signal pathway in NMBA-induced rat tumorigenesisObjective: To study the effect of TCCP on wnt signal pathway in NMBA-induced rats tumorigenesis and to investigate the mechanism by which TCCP modulate tumorigenesis. Methods: Total cellular RNA was isolated from frozen tissues using TRIzol Reagent. The expression of c-myc mRNA was detected by RT-PCR. Western blot was used to investigate the protein expression of GSK-3βandβ-catenin in the esophageal epithelium.Results: 1 The gene expression of c-myc of esophageal epithelium in NMBA control group was significantly increased at weeks 9, 15, 25 compared with normal control (P<0.05). TCCP supressed the mRNA expression of c-myc at both weeks 9 and 15 (P<0.05), but not at weeks 25.There was no significant difference between the high TCCP group and low TCCP group.2 It was observed that the expression ofβ-catenin was slightly present in normal mucosa of esophagi of the rats which were untreated with NMBA, and then was significantly increased by administering NMBA from weeks 9 to 25 (P<0.05). The up-regulatedβ-catenin expression was decreased significantly by TCCP treatment at each check-point (P<0.05). The expression of GSK-3βwas rich in normal mucoa of esophagus, but was decreased significantly by administering NMBA, and was lowest at 25th week (P<0.05). The decreased protein levels of GSK3βwas significantly elevated by TCCP treatment at each check-point (P<0.05). But there was no significantly between the two treatment groups.Conclusions: TCCP inhibited NMBA-induced rat esophageal carcinogenesis probably via acitivition of GSK-3β, suppression ofβ-catenin and c-myc expression.Part 3 Effects of triterpenes compound of Cortex Periplocae on cyto-immunologic function in N-nitrosomethylbenzylamine-induced esophageal tumorigenesis in F344 ratsObjective: To investigate the mechanism and effects of triterpenes compound of Cortex Periplocae on cyto-immunologic function in N-nitrosomethyl- benzylamine-induced esophageal tumorigenesis in F344 rats.Methods: 1 FCM and RT-PCR were used to detected the proportion of CD4+CD25+ and gene expression of Foxp3 in peripheral blood of rats . 2 RT-PCR and ELASA were used to detected the gene and protein expression of IL-2, IL-10 and TGF-β1 in peripheral blood of rats .Results: 1 At week 9 after the NMBA treatment, CD4+T cells in peripheral blood of NMBA group rats were decreased significantly and CD4+CD25+ regulatory T cells and Foxp3 mRNA in peripheral blood of NMBA group rats were significantly increased, which deteriorated further with the time going on (P<0.05). At weeks 9 and 15, TCCP treatment can decreased the proportions of CD4+CD25+ regulatory T cells and the gene expression of Foxp3, while the proportions of CD4+ cells were significantly increased (P<0.05), compared with NMBA group. At week 25, TCCP significantly decreased the expression of Foxp3 mRNA, but had no significant effect on CD4+CD25+Treg and CD4+T cell.At week 9 after NMBA treatment, the protein levels of TGF-β1 and IL-10 in peripheral blood of in F344 rats were significantly higher than those in normal control group and soy control group. The gene expression of TGF-β1 and IL-10 were also significantly elevated. While the levels of protein and gene expression of IL-2 were significantly decreased, as compared with normal control. TCCP can decrease the expression of IL-10 and TGF-β1, increase the expression of IL-2.Conclusions: The expression of CD4+CD25+ regulatory T cells and Foxp3 mRNA in peripheral blood of rats were enhanced after treatment with NMBA, while the triterpenes compound of Cortex Periplocae can effectively inhibit the expression, thus it obviously improve the immune dysfunction in experimental esophageal tumorigenesis in F344 rats.
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