| Background:Colorectal cancer is the most frequent cancer in our country, and its morbility is very high. Following the development of economy, the increase of people's living standard and the changes of life style and dietary pattern in our country, the morbility of colorectal cancer has gradually risen year by year. There are many kinds of carcinogens and mutagens included in heterocyclic amine (HACs) in the cooked meat. The compound of HCAs mostly exists in toasted (or baked) meat and fish. People are nearly exposed in mutagens (or carcinogens) HACs every day. Epidemiological researches have indicated that when people have intaken a quite large amount of over ripening meat, they will have more possibility to suffer from colorectal cancer than other people. Among the HCAs, 2-Amino-3-methylimidazo [4, 5-f] quinoline (IQ) is relative major in content. IQ can cause gene mutation and microsatellite instability in colorectal cancer, thus it has obvious carcinogenesis.Although the researches on colorectal cancer have been made great progress, status quo of high morbidity and mortality has not been changed. At present, because of the utilized convenience of and having no harm of food, chemical substance preventing cancer in food has become the hot spot of the research, such as diferuloylmethane, raphanin and licopersicin. The preventive effects of these chemical substances lie in the following four aspects. The first effect of these chemical substances is to prevent the formation of some carcinogens in human body. The second is to activate the protein molecules in cells and wrap the carcinogens intruding into the body. Then the carcinogens are discarded out of the body. So the injury of carcinogens towards cell nuclei is prevented, and the integrity of gene is retained. The third is to eliminate the incipient foci of cancer and inhibit the growth of blood capillary in the domain of canceration. Thus the cells in the domain of canceration will atrophy to die of hypoxia and defective nutrition. The last effect of these chemical substances is to facilitate the formation of lymphocyte in human body, which can take part in the struggle against cancer in body.There has been the effect of health maintenance in record from ancient. Tea polyphenols, the whole name of more than 30 polyphenols in tea, is a pure natural mixture extracted from green tea. It is mainly composed of four materials in big category, and catechin is the most important among them. It accounts for 60-80% in polyphenols. Catechin mainly includes several monomers such as EGC, EGCG, EC, and ECG and so on. Among all the polyphenols, EGCG is most abundant. Some researches have shown that EGCG can inhibit the proliferation and promote the apoptosis of cancer cells in vitro and in vivo, and also induce the express of phase II drug metabolizing enzymes in intestinal tract. So EGCG has the effect to prevent carcinogenesis.Uridine 5'-diphosphate-glucuronosyltransferase (UGTs) is the most important phase II drug metabolizing enzymes when the chemical substances are undergoing the biological transformation. It mediates the combination of glucuronic acid and many chemicals, increases their water-solubility, and excretes them in urine and bile. Although liver microsome is the major site of combination between glucuronic acid and other materials, the isoforms of UGT1A8 and UGT1A10 are expressed exclusively in gastrointestinal tract. The combination of glucuronic acid and other substances in gastrointestinal tract is a supplement to the detoxication of liver, and educes the metabolism barrier function of the mucosa in gastrointestinal tract.Nuclear transcription factor erythroid 2p45 (NF-E2)-related factor 2 (Nrf2), one member of the family of bZIP transcription factors, has relationship with the induction of metabolizing enzymes especially phase II drug metabolizing enzymes. The induction of phase II enzyme genes is regulated by their as-acting antioxidant response element (ARE) or electrophile responsive element (EpRE). The transcriptional factor of Nrf2 binds to ARE/EpRE and regulates transcription through them after heterodimerizing with one of the small Maf proteins. Some agents exert cancer chemoprevention by strong induction of phase II enzymes including UGTs via activation of Nrf2.The present research is to investigate whether EGCG has the preventing effect against the formation of aberrant crypt foci (ACF) and the histological alteration induced by IQ in colonic mucosa in immunologic deficit mice. And we are going to analyze the expression of mRNA and protein of Nrf2 and UGT1A10. We will construct orthotopic colon cancer models in nude mice with HT-29 cell line and HT-29-siNrf2 cell line. After different doses of EGCG are administrated to nude mice by oral gavages, we observe the growth and metastases of colon cancer. Besides, we will observe the mRNA and protein expression of the genes of Nrf2, UGT1A, UGT1A8 and UGT1A10, and further analyze the correlated mechanism of EGCG preventing colon cancer.Objectives:1. The first experiment is to observe the influence of different doses of EGCG on the formation of ACF induced by IQ in colon of nude mice, and observe histological alteration of colon. In addition, it is to analyze the mRNA and protein expression of Nrf2 and UGT1A10, and thereby investigate possible molecular mechanisms of EGCG preventing preneoplastic lesions in colon.2. The second experiment is to construct subcutaneously cancer model with HT-29 cell line in nude mice. Using the cancer tissue growing subcutaneously, we will construct orthotopic colon cancer model in nude mice. We will observe the inhibition effects of different doses of EGCG on the growth, peritoneum metastases, the ascites formation, and liver and pulmonary metastases. We will also analyze the mRNA and protein expression of the genes of Nrf2, UGT1A, UGT1A8 and UGT1A10in cancer tissue. Thereby we investigate whether EGCG has preventive effects on the growth and metastases of colon cancer through inducing the expression of UGT1A and its isforms.3. Blocking the expression of Nrf2 in HT-29 cell line in vitro with the technology of RNA interference, we construct subcutaneously cancer model in nude mice with HT-29-siNrf2 cell line. Using the cancer tissue growing subcutaneously, we will construct orthotopic colon cancer model in nude mice. We will observe the inhibition effects of different doses of EGCG on the growth, peritoneum metastases, the ascites formation, and liver and pulmonary metastases. We will also analyze the mRNA and protein expression of the genes of Nrf2, UGT1A, UGT1A8 and UGT1A10in cancer tissue. Thereby we investigate the role of Nrf2 playing in the EGCG inducing the expression of UGT1A and its isforms.Methods:1. We constructed an animal model with carcinogen IQ to induce the formation of ACF in colon of nude mice. 0.2% Methylene blue staining was used to observe the number of ACF and AC in mice after administrated with low-, medium- and high-dose EGCG (5, 10 and 20mg/kg.d). H&E staining was performed for histopathological observation in colon mucosa. The protein expression of Nrf2 was detected by IHC and western blotting in colon. The mRNA levels of Nrf2 and UGT1A10 were detected by RT-PCR. At last we analyzed the relation of the formation of ACF and the expressions of Nrf2 and UGT1A10.2. We routinely cultivate the HT-29 cell line to logarithmic phase in vitro. The next is to construct subcutaneously cancer model with HT-29 cell line in nude mice. After having been passaged for five generations, the tumor growing subcutaneously was resectted. Using subcutaneous tumor tissue we construct orthotopic colon cancer model in nude mice. After mice having been administrated with low-, medium- and high-dose EGCG (5, 10 and 20mg/kg.d), we observed if there were peritoneum metastases and ascites formation with our naked eye. And we observed liver and pulmonary histological alteration with H&E staining. The protein expression of Nrf2 was detected by IHC and western blotting in colon cancer tissue. The mRNA levels of Nrf2, UGT1A, UGT1A8 and UGT1A10 were detected by RT-PCR. We analyzed the inhibition of different doses of EGCG on the growth and metastases of colon cancer, and the induction of Nrf2, UGT1A, UGT1A8 and UGT1A10.3. We used the technology of RNA interference to blocking the expression of Nrf2 in HT-29 cell line in vitro, we construct with low expression of Nrf2. We cultivate the cell line to logarithmic phase. Using HT-29-siNrf2 cell line we construct subcutaneous cancer model in the nude mice. After having been passaged for five generations, the tumor growing subcutaneously was resectted. Using subcutaneous tumor we construct orthotopic colon cancer model in nude mice. After mice having been administrated with low-, medium- and high-dose EGCG (5,10 and 20mg/kg.d), we observed if there were peritoneum metastases and ascites formation with our naked eye. And we observed liver and pulmonary histological alteration with H&E staining. The protein expression of Nrf2 was detected by IHC and western blotting in cancer tissue. The mRNA levels of Nrf2, UGT1A, UGT1A8 and UGT1A10 were detected by RT-PCR. We analyzed the inhibition of different doses of EGCG on the growth and metastases of colon cancer. Therefore the role of Nrf2 in inducing the expression of UGT1A, UGT1A8 and UGT1A10 caused by different doses of EGCG was proved from reverse direction.4. Statistical analysis: The statistic software of SPSS13.0 for windows was used to analyze the significance of the differences. Measurement data were presented as means±SD. The significance of the differences among four groups was analyzed by means of One-Way ANOVA. Enumeration data were presented as percentages. The means of Chi-square test was used to analyze the significance of these differences. P-values less than 0.05 were considered statistically significant.Results:1. The results of the first part were that the body weights of mice in IQ group decreased significantly, high degree atypical hyperplasia appeared and a large amount of ACF formed in colon mucosa. After administrated different doses of EGCG in mice of three EGCG groups, the body weights of mice increased significantly, and histological alteration in colon tended to normal. The number of ACF reduced obviously. The protein expression of Nrf2 detected by IHC and western-blotting in colon increased significantly compared with that of mice in IQ group (P<0.05), and there was a phenomenon of nuclear transcription of Nrf2. The mRNA levels of Nrf2 and UGT1A10 detected by RT-PCR were higher than those of mice in IQ group (P<0.05).2. The results of the second part were that there were several mice with orthotopic colon cancer formed by HT-29 cell lines appeared dyscrasia and athrepsy. After administrated different doses of EGCG, the body weights of mice in three EGCG groups increased, and the growth and multiple metastases (including peritoneum, abdominal cavity, live and pulmonary) of orthotopic colon cancer were inhibited. The protein expression of Nrf2 detected by IHC and western-blotting in colon increased significantly compared with that of mice in model group (P<0.05), and there was a phenomenon of nuclear transcription of Nrf2.The mRNA levels of Nrf2, UGT1A, UGT1A8 and UGT1A10 detected by RT-PCR were higher than those of mice in model group (P<0.05).3. The results of the third part were that the body weights of the mice in four groups with orthotopic colon cancer formed by HT-29-siNrf2 cell lines had no significant difference. After administrated different doses of EGCG, the growth and multiple metastases (including peritoneum, abdominal cavity, live and pulmonary) of orthotopic colon cancer in four groups had no significant difference (P>0.05). The protein expression of Nrf2 detected by IHC and western blotting in colon had no significant difference compared with that of mice in model group (P>0.05). The mRNA levels of Nrf2, UGT1A, UGT1A8 and UGT1A10 detected by RT-PCR had no significant difference compared with those of mice in model group (P>0.05).Conclusions:1. Different doses of EGCG could inhibit the formation of ACF in colon of nude mice. So EGCG could prevent the occurrence of colon cancer and had dose-dependent effect. This anti-cancer effect could possibly owe to the increase of the IQ elimination in colon through inducing the expression of Nrf2 and UGT1A10 and the reduction of the ACF formation.2. Different doses of EGCG could inhibit the local growth and metastases of orthotopic colon cancer in nude mice. And this inhibition had dose-dependent effect. The inhibition effect was caused by inducing the expression of the genes of Nrf2, UGT1A, UGT1A8 and UGT1A10. Thus EGCG could increase the resistivity of colonic mucosa against colon cancer (including local growth and distant metastases).3. Compared with model group (orthotopic colon cancer constructed with HT-29-siNrf2 cell line in nude mice) , different doses of EGCG had no significant inhibition on the local growth and distant metastases and did not induce the expression of Nrf2, UGT1A, UGT1A8 and UGT1A10 obviously. This indicated that Nrf2 played a critical role in EGCG inducing the expression of UGT1A and its isforms.Significance:1. The first part has proved that different doses of EGCG can inhibit the formation of ACF induced by IQ in colon, and constructed an animal model of phytochemistry protective agents against colorectal cancer. This result has established the experimental basis for the research on the prevention against colorectal cancer in vivo.2. The second part has proved that different doses of EGCG can inhibit the local growth and multiple metastases of orthotopic colon cancer in nude mice. And it is the first time that EGCG has been presented to induce the expression of the Nrf2-UGT1A signal pathway in the model of orthotopic colon cancer. This result has established the experimental basis for simulating the growth and metastases of human colon cancer in nude mice, and investigating the new mechanism of the chemoprevention against colon cancer.3. The third part has further proved from reverse side that EGCG can induce UGT1A and its isforms. It is nuclear factor Nrf2 that plays a role of bridge. This result has established the experimental basis for the mechanism of Nrf2 participating the induction of metabolizing enzymes in vivo.
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