| Paper onePolymorphic expression of UDP-Glucuronosyltransferase UGT2B gene in human colorectal epitheliumBackground: The UDP-Glucuronosyltransferases (UGTs) are phase II biotransformation enzymes that glucuronidate numerous endobiotic and xenobiotic substrates. Glucuronidation increases the water solubility of the substrate and facilitates renal and biliary excretion of the resulting glucuronide conjugate. UGTs have been divided into two gene families, UGT1 and UGT2. There are marked interindividual differences in the content of UGTs in the liver and other organs. In general, glucuronidation serves an inactivating or protective function by terminating or attenuating the biological activity of its substrates. UGT superfamily can be devided into 2 family, UGTl family involved in metabolism of thyroid hormones, phenol and bilirubin, UGT2 family involved in metabolism of bile acid, steroid and BaP. Human know littlee about UGT2B, so we do some work on the expression of UGT2B in human colorectal epithelium, to detect the effect of UGT on colorectal carcinogensis.Objective: To detect the expression of UDP-Glucuronosyltrans -ferase (UGT) 2B subfamily in colorectal cancer, and to investigate itssignificance in colorectal tissue carcinogenesis.Methods: Reverse transcription and Polymerase chain reaction (RT-PCR) were used to determine the mRNA levels of four UGT isoforms and transcription factors Hepatocyte Nuclear Factor 1 α in 32 specimens of colorectal cancer, 14 specimens of normal colorectal tissues.UGT2137 and Hepatocyte Nuclear Factor 1 α protein was detected using western blot analysis, indirect immunofluorescence analysis was performed for detection of colonic UGT2B7 protein in situ.Results:Polymorphic regulation of UGT2B isoforms expression in human colorectal tissue, Analysis of the colorectal cancer tissue samples revealed UGT2B4 expression in 25 of the 32 samples, UGT2B7 was expressed in 21 of the 32 samples, UGT2B15 was expressed in 11 of the 32 samples. Compared to control group, UGT2B4 mRNA level of the 32 samples of colorectal cancer and its surrounding healthy tissue was not significantly changed; UGT2B7, UGT2B15 mRNA level was significantly down-regulated in colorectal cancer and its surrounding healthy tissue; HNF1 α mRNA level of the 32 samples of colorectal cancer and its surrounding healthy tissue was not significantly changed;In colon, UGT2B7 immunofluorescence was localized in the epithelial cells of mucosa only, an immunofluorescence pattern excluding nuclei with accumulation in the perinuclear space was noted within the cells of both tissues was comparable, but the staining of the individual cancer cell is lower than that of the normal colon epithelium cell.UGT2B7 protein was detected by western blot analysis, the odds of the protein was obtained from bandscan 5.0 Demo. Compared to control group, UGT2B7 protein level was significantly down-regulated in colorectal cancer and its surrounding healthy tissue. The analysis of pairs of tumor tissue and its surrounding healthy tissue demonstratedlower levels of UGT2B7 protein in the colorectal cancer tissue(0. 186± 0. 109vs0. 409 ± 0.171, P<0.01 ,while the odds was 0.684 ± 0.117 in colorectal tissue of normal persons. Conclusion:UGT2BmRNA expression and protein expression level were decreased in colorectal cancer tissues and its surrounding healthy tissue. This implied that interindividual regulatory mechanisms may be the biological basis that determines interindividual variations in cancer risk. The mRNA and protein level of Hepatocyte Nuclear Factor 1 α (HNF 1 α ) were not changed, may be there have complicated regulatory mechanisms.Paper twoEffects of bile acids on HNF-1 α and UGT of human colorectal cancercell line Caco -2Background: A great deal of research were done about relation between bile acids and colorectal cancer in domestic and international. The Epidemiology show that the Euro-American crowd of the high fat, high egg white, low cellulose food, the outbreak rate of the colorectal cancer is obvious high in the person of Asia and African persons of the low fat, the high cellulose food, and bile acid is the main medium thing of the high fat, high egg white food and colorectal cancer, also is to say that the food promotes bile acid secreted sourly, the latter promotes tumor take place. The UDP-Glucuronosyltransferases(UGTs) are phase II biotransformation enzymes that glucuronidate numerous endobiotic and xenobiotic substrates including bile acids and mutagens. Glucuronidation increases the water solubility of the substrate and facilitates renal and biliary excretion of the resulting glucuronide conjugate. The UGT is mainly to express in the liver, but extrahepatic, also having the expression in an organization of stomach and intestines particularly, becoming the barrier of GI tract mucosa, along with the bowel way from the near to the distant, express the quantity to descend gradually, this be the also likely to be reason that distant place colorectal alters to take place the tumor.Objective: This part inquiries into detect the effect of bile acids on the UGT expression and proliferative activity of colorectal cancer cell Caco -2, into the mechanism of its tumor growth.Methods: MTT assay were used to detect the proliferative affect of CA, DCA on colorectal cancer cell Caco — 2. Reverse transcription and Polymerase chain reaction(RT-PCR) were used to determine the mRNA levels of four UGT isoforms and transcription factors Hepatocyte NuclearFactor 1α in colorectal cancer cell Caco —2 effected by primary bile acid CA, second bile acid DCA and control .UGT2B7 and Hepatocyte Nuclear Factor 1 α protein was detected using western blot analysis and immunocytochemistry.Results:There are no significant different in the mRNA expression of UGT1A1, UGT1A6, UGT2B4and UGT2B7 between CA treatment group and control ( P>0.05) , the colorectal cancer cell Caco-2 were harvested after it were treated for 24h. CA group have no significant proliferative than control.There are no significant different in the mRNA expression of UGT1A1,UGT2B4 between DCA treatment group and control(P>0.05) the colorectal cancer cell Caco-2 were harvested after it were treated for 24h. DCA promote the mRNA expression of UGT1A6, and decrease the mRNA expression of UGT2B7 in dose-dependent and time- dependent pattern. Among the research of DCA effect on the mRNA expression of UGT1A6, UGT2B7, there is apparent change after drug treat for 24h, and it arrived the highest after treatment for 48h.There are no significant different in the mRNA expression of HNFl a between CA treatment group and control ( P>0.05) ; Compared to control, 25μmol/l, 50μmol/l DCA treated group have no apparent change, 75μmol/l,100μmol/l DCA treated group decrease the mRNA expression of HNF1α in significant manner.Treated with DCA for 24h produced a significant decrease in UGT2B7 protein band intensity in Caco-2, compared to control group. Immunocytochemistry analysis revealed that there is significant change of HNF1α protein level between DCA treatment group and control.Conclusion: 1. There are no significant proliferative effect of CA on the colorectalcancer cell Caco-2, DCA can promote the growth of the colorectal cancer cell Caco-2.2. There are no significant different in the mRNA expression of UGT1A1, UGT1A6, UGT2B4and UGT2B7 between CA treatment group and control; DCA increase the mRNA expression of UGT1A6, and decrease the mRNA expression of UGT2B7 in dose-dependent and time-dependent pattern.3. There are no significant different in the mRNA and protein expression level of HNF1 α between CA treatment group and control, DCA can decrease the mRNA and protein expression level of HNF1α than control.Paper threeEffects of TBHQ on HNF-1α and UGT of human colorectal cancercell line Caco -2Background: The application of antioxidant-type inducers gain more and more value in the chemistry prevention strategy of the colon cancer. Chemopreventive induction mechanisms are known to be triggered by a large variety of constituents of our plant diet, including polyphenolic flavonoids in onions such as quercetin, organosulfur compounds in garlic, and isothiocyanates in broccoli . These adaptive mechanisms probably evolved as a consequence of feeding on a plant diet. Hence, electrophilic metabolites derived from dietary plant constituents appear to trigger an adaptive response that enhances the antioxidant defense, the latter including ancillary enzymes such as UGTs. The enzymes induced by antioxidant-type inducers (UGTs, NQO1, and GSTs) may be key players in the detoxification.Colorectal cell line Caco-2 is a good model for UGT inductive research, as it come from human colorectal cell, have the same morphic and enzymic characteristics, it can form epithelium structure with good tight connection. The Caco-2 cell model appears, therefore, to be useful to identify the DNA domain(s) responsible for antioxidant-type induction of UGTs. three human UGTs (UGT1A6, UGT1A9, and UGT2B7) are inducible by antioxidant-type inducers and at least two UGTs (UGT1A6 and UGT1A9) are inducible by AhR agonists TCDD .Objective: In this study, we cultured Colorectal cell line Caco-2 and investigate whether UGT can be induced by TBHQ, and TBHQ affect the proliferative activity, colon forming ability, morphological variations under transmission electron microscopy and cell cycle analysis of Caco-2.Methods: MTT assay were used to detect the proliferative affect of TBHQ on colorectal cancer cell Caco —2. Morphological variations were observed under transmission electron microscopy and cell cycle analysis of Caco-2 were detected by Flow cytometry analysis. Reverse transcription and Polymerase chain reaction(RT-PCR). were used to determine the mRNA levels of four UGT isoforms and transcription factors Hepatocyte Nuclear Factor 1α in colorectal cancer cell Caco —2 effected by TBHQ and control .UGT2B7 and Hepatocyte Nuclear Factor 1 α protein was detected using western blot analysis and immunocyto-chemistry. Results:1. Effect of TBHQ at various concentrations on the growth of Caco-2 cells: TBHQ produced a dose-dependent and time-dependent inhibition of cell growth.2. Colony forming ability assay showed significant inhibition in Caco-2 treated with TBHQ, and indicating dose-dependent manner.3. Under Transmission eletron microscopy, control cells were big and round, with intact nuclear membrane and low density in nuclear chromatin. However, the cells treated with TBHQ exhibited characteristics of apoptosiss, and cell membrane shrinkage, cytoplasm budding, condensation and fragmentation of nuclear chromatin adjacent to nuclear membrane.4. Flow cytometry analysis revealed G2/M phase accumulation and decreasing GO/Gl phase cell ware caused by TBHQ treatment.5. There are no significant different in the mRNA expression of UGTIAK UGT2B4 between TBHQ treatment group and control ( P> 0.05) the colorectal cancer cell Caco-2 were harvested after it were treated for 24h. TBHQ promote the mRNA expression of UGT1A6, and increase the mRNA expression of UGT2B7 in dose-dependent and time- dependent pattern. Among the research of TBHQ effect on themRNA expression of UGT1A6, UGT2B7, there is apparent change after drug treat for 24h, and it arrived the highest after treatment for 48h. Treated with TBHQ for 24h produced a significant increase in UGT2B7 protein band intensity in Caco-2, compared to control group. Inimunocytochemistry analysis revealed that there is significant increase of HNF1α protein level between TBHQ treatment group and control. Conclusion:1. There are significant antiproliferative effect of TBHQ on the colorectal cancer cell Caco-2 in a dose-dependent and time-dependent manner.2. TBHQ promote the mRNA expression of UGT1A6, and increase the mRNA expression of UGT2B7 in dose-dependent and time- dependent pattern.3.TBHQ can increase the mRNA and protein expression level of HNF1α than control.
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