Clinical And Basic Research On Expression Of UGT1A Gene Locus And Induction Of Sulforaphane In Colorectal Cancer

Paper onePolymorphic expression of UDP-glucuronosyltransferase UGTIA gene locus in human colorectal epitheliumBackground:The glucuronosyltransferases (UGT) play a critical role in phase II metabolism.They catalyze the glucuronidation of N-hydroxy compounds and prevent DNA mutation evoked by heterocyclic amines.The colon mucosa shows the most complex polymorphic expression of UGTIA gene locus, especially the identification of UGT1A8,UGT1A1O expression in colorectal epithelium emphasizes a unique physiological role of glucuronidation in genoprotection.In view of the significant role of UGTIA isoforms in metabolism and elimination of the potential carcinogens ,as well as the regulation in a tissue specific fasion,we investigated UGT1A gene locus expression and enzyme catalysis in colorectal cancer and tried to find the discrepancy between the cancer tissue and the normal colorectal tissue.We also focused on the problem different predisposition of colorectal cancer derived from the differential UGTIA gene expression. To study the polymorphic regulation , UGT1A mRNA expression and UGT catalytic activities were investigated by RT-PCR,Western blot analysis and HPLC.Objective: To analyze the polymorphic expression of the glucuronosyltransferase UGTIA gene locus in human colorectal epithelium, and reveal the significant role of the polymorphic regulation in the colorecatal cancer.Methods: Colorectal tissue samples were obtained from 40 patients with colorectal cancer and 20 normal person controls,and normal liver tissue samples were collectedfrom 8 patients with hepatic cyst and hemangioma.The panel included 40 tissue sample pairs,each consisting of a sample of tumor tissue and a sample of the surrounding healthy colorectal mucosa. UGTIA transcription were performed by RT-PCR and exon-1 specific RT-PCR, and UGTIA protein was detected using western blot analysis, indirect immunofluorescence analysis were performed for detection of colonic and hepatic UGT1A protein in situ. Meanwhile catalytic activity assay of microsomes was characterized using N-hydroxy-PhIP as substrate.Statistical analysis(student t test and one-way ANOVA) were carried out using SPSS 11.0 software package. Results:(1)Quantitative variability of UGTIA mRNA.Overall UGTIA mRNA expression was lower in colorectal tissue of normal person compared with hepatic tissue (0.916±0.111 vs 1.219±0.126, P<0.01) .Between the 40 samples of colorectal cancer, UGT1A mRNA expression was found to be significantly down-regulated in colorectal cancer tissue compared to the surrounding healthy tissue (O.279±O.215vsO.688±O.326, P<0.01 ) ,while the latter was lower than that of colorectal tissue of normal person(P<0.01).Analysis of UGT1A mRNA in the individual tissue samples also indicated the colorectal UGTIA gene expression was characterized by significant interindividual variation compared with the more uniform UGTIA mRNA found in hepatic tissue.Polymorphic regulation of UGT1A isoforms expression in human colorectal tissue.Analysis of the surrounding healthy colorectal tissue samples revealed UGT1A8 expression in 33 of the 40 samples,UGT1A10 was expressed in 32 of 40,whereas UGT1A1 was identified in 25 samples,UGT1A3 in 18 samples, UGT1A4 in 11 samples,UGT1A6 in 9 samples,and UGT1A9 in 16 samples.The expression of UGT1A5 and UGT1A7 was not detected in any of the tissues.The patterns identified in the healthy colorectal tissue specimens were confirmed in the corresponding cancer tissue samples.In these samples ,UGT1A8 were expressed in 39 of 40 samples, UGT1A10 were expressed in 38 of 40 samples,while UGT1A1 in 23 of 40 samples, UGT1A3 in 15 of 40 samples ,UGT1A4 in 11 of 40, UGT1A6 in 5 of 40 andUGT1A9 in 5 of 40 samples.The polymorphic expression is present in the colorectal tissue of normal persons,while the analysis of 8 hepatic tissue displayed no polymorphic expression.UGT1A1,UGT1A3,UGT1A4,UGT1A6,UGT1A9 mRNA levels were differentially down-regulated in the tumor tissue compared with surrounding normal mucosa(P<0.01) ,in contrast,UGTlA8 and UGTIAIO showed up-regulation(P<0.01), whereas UGTlA8 and UGTIAIO mRNA expressions was lower than that of the two isoforms gene expression of colorectal tissue of normal person.(2) Overall UGTl A protein was detected by western blot analysis,the odds of the protein was obtained from bandscan 5.0 Demo .The analysis of pairs of tumor tissue and the surrounding healthy tissue demonstrated lower levels of overall UGTl A protein in the colorectal tumor tissue(0.235±0.101vs0.524±0.135.P<0.01),while the odds was 0.665±0.212 in colorectal tissue of normal persons,and 0.701±0.006 in hepatic tissue.The finding is in agreenment with the results obtained by RT-PCR on the mRNA level. In colon, UGTl A immunofluorescence was localized to the epithelial cells of the mucosa only, and in liver homogeneous staining was observed throughout the lobuluso .An immunofluorescence pattern excluding nuclei with accumulation in the perinuclear space was noted within the cells of both tissues, the staining within the individual cells of both tissues was comparable,but the staining of the individual cancer cell is lower than that of the normal colon epithelium cell.(3) In both colon and human liver, the predominant glucuronide of N-OH-PhIP is the N-OH-PhBP-N2-glucuronide. Microsomal glucuronidation activities were examined ,and the tumor tissue showed glucuronidation activity as 231±12 pmol/min/mg, the surrounding healthy tissue showed as 435±24pmol/min/mg,while the latter activity was lower than that of the colonic tissueof the normal person(P<0.01). There was considerable interindividual variability between the samples in the ability to glucuronidate N-OH-PhIP. Conclusion:(1) Human UGTl A genes are regulated in a tissue-specific fashion in colorectal epithelium and hepatic tissue.(2) Polymorphic expression of UGT1A gene locus is present in both tumor tissue and the surrounding healthy tissue,also in colorectal tissue of normal persons.While it displayed no polymorphism UGT1A expression in human liver.(3)These data provided evidence for the polymorphic regulation of UGT1A gene locus at the RNA transcript and functional levels in colorectal epithelium,and this also implied that interindividual regulatory mechanisms may be the biological basis that determines interindividual variations in cancer risk. Paper twoPolymorphism of the human UDP-glucuronosyltransgerase UGT1A8 genein colorectal cancerBackground: The human UDP-glucuronosyltransferase(UGT) belongs to a superfamily of nine proteins encoded at the human UGT1A gene locus on chromosome 2q37. UGTs catalyse glucuronidation which represents a metabolic detoxification ,and target substrates for glucuronidation consist of compounds from many divergent chemical classes, including dietary byproducts, endogenous metabolites such as steroid and bilirubin, many therapeutic drugs, and many environmental mutagens,such as heterocyclic amines as well as polycyclic aromatic hydrocarbons.The gene encoding UGT proteins is polymorphic.UGTlA8 is mainly expressed in colorectal tissue,and the gene missense mutations in exon 1 lead to amino acids substitution which reduce catalytic activity.These finds prompted us to predict that alterations in UGT1A8 gene expression or in the functional properties of UGT1A8 may be indicators of potential cancer risk.Objective:We examined UGT1A8 as a target gene for the evaluation of polymorphism and their relation to colorectal cancer.Methods: 69 cases of colorectal cancer and 69 controls were collected for analysis.They were han people from Shandong province and their ages difference were less than 5 years old.Genomic DNA was prepared from full blood samples. Using exon-1 specific primer, we performed PCR amplification of UGT1A8 exon-1 sequences.And the products were visualized by gel electrophoresis and the fragments were puried .When needed,the extracted DNA was subcloned into TOPO TA cloning vectors and the insert sequenced for identification of allelic specificity.Statistical analysis (two tailed Fisher's exact test,odds ratios ORs confidence interval CI)was carried out using SPSS 11.0 software for all identified genotypes ,the combined UGT1A8*3 allelic genotypes.Results:(1 identification of the UGT1A8 alleles:Three mutations in UGT1A8 exon 1were identified. Base pair 518C>G, base pair 765A>G and base pair 830G>A. The 518 mutation led to a missense mutation in codon 173 resulting in an alanine being substituted for a glycine ,while the 830G>A mutation in codon 277 resulted in a cysteine conversion to a tyrosine. The mutation at base pair 765A>G is silent.(2)Identification of the UGT1A8 alleles frequencies and genotyes.UGTlA8 polymorphisms were found in colorectal cancer cases and controls.Within controls, 51.5% alleles were identified as wild UGT1A8*1 (including UGTlA8*la),while 32.6% in cases, P=0.047, indicating the discrepancy being significant.ORs 0.51(95% CIO.28-0.79) predicted this allele was an protective factor.The identity of UGT1A8*3 was indeed rare/with only 2.2% in controls, while 15.9% in cases( P=0.003). OR 9.63 (95% CI2.12-7.79) indicated UGT1A8*3 as a risk factor of cancer.a homozygous pattern of UGT1A8*1 expression(genotypes UGT1A8*1/*1, UGTlA8*l/*la, UGTlA8*la/*la) was observed in 34.78% of this population in controls, 13.04% in cases(P=0.045).The heterozygous expression of UGTlA8*l/*3 was 2.90% and 13.04% in controls and cases respectively, P=0.028, OR 5.03(95% CI 2.25-5.99); The heterozygous expression of UGTlA8*2/*3 was 1.45% and 15.94% in controls and cases respectively, P=0.003, OR12.90(95% CI2.68-6.15) .These findings indicated the two genotypes were major risk fator of colorectal cancer.Logistical regression analysis demonstrated that tumor occurance was dependent on the presence of polymorphic UGT1A8 alleles (P=0.029),in particular UGT1 A8*3 (P=0.001) ,but was independent of sex(P=0.25).Conclusion:(l)The high incidence of UGT1A8 polymorphism detected among the patients with colorectal cancer was remarkerble, UGT1A8*1 allele was a protective factor and UGT1A8*3 allele was a risk factor for CRC.(2) homozygous pattern of UGTlA8*l/*3^ UGTlA8*2/*3 were also associatied with the high occurance of CRC. Paper threeExperimental investigation on induction of UDP-glucuronosyltransferase UGT1A by sulforaphane in Caco-2 cellBackground:Our prophase research indicated that UDP-glucuronosyltransferas UGT1A gene expression and microsomal activities were down-regulated in the colorectal epithelium. On account of the detoxification of the carcinogen and the expression in tissue-specific fashion of UGT1A and the isoforms UGTlAl,UGTlA8,UGTlA10,we think that the lower expression of UGT1A gene and lower activities of microsomal protein may be one of the mechanism in colorectal cancer. The consumption of cruciferous vegetables has a protective effect on the development of colorectal cancer. The phytochemical sulforaphane is an isothiocyanate found almost exclusively in cruciferous vegetables and can be potent inducers of phase II detoxification enzymes, such as glutathione S-transferase (GST).Therefore we had consideration for the induction of UGT1A and the isoforms by the phytochemicals and facilitated glucuronidation of heterocyclic aromatic amine to decrease the risk of colorectal cancer.Objective: In this study,we cultured human colon cancer cells Caco-2 in vitro and determined the specific UGT1A isoforms induced and whether this induction facilitied glucuronidation and potential detoxification of the colon carcinogen N-hydroxy-PhlP.Meanwhile we also investigated the signal transduction mechanism of the induction.Methods: A human colon cancer cell line Caco-2 was cultured in vitro ,and cells in logarithmic growth phase were selected and examined.(1) The antiproliferative affect of sulforaphane of lO-lOOtoiol/L at concentration was examined by means of MTT assay ,and IC50 calculated.(2) The UGT1A mRNA expression were measured by RT-PCR,which were inducted by sulforaphane of 5jxmol/L^ 10umol/I^ 15|j,mol/L^ 20ujtnol/L^ 25umol/I^ 30u.iik)1/Ln 35umol/L at concentration below IC50.(3) UGT1A protein levels were determined by western blotting,and glucuronidation rates of N-hydroxy-PhIP were showed by high performance liquidchromatography(HPLC).(4)To study the molecular biological mechanism of induction , we measured Nrf2 mRNA expression performed by RT-PCR,and observed the nuclear localization of transcription factor Nrf2 by confocal laser microscope.(5) Statistical analysis(student t test ,one-way ANOVA and pearson correlation analysis) were carried out using SPSS 11.0 software package.Results:(DAfter cells were treated with sulforaphane of various concentration for 24h,significant increase in UGT1A mRNA was observed at 10|amol/L-35u.mol/L concentration with no cytotoxicity(P<0.05).The induction of sulforaphane was in concentration -dependent manner,and the highest induction occurred at a half of IC5o(25urnol/L).Meanwhile we observed the induction with 15umol/L sulforaphane increased in time-dependent manner.UGTlAl,UGTlA8,UGTlA10 mRNA levels are significantly increased in the cells treated with 15umol/L sulforaphane compared to the controls (P=0.006, P=0.017, P=0.008,respectively).(2) Treated with sulforaphane(10umol/L-30urnol/L) for 24h produced a significant increase in UGT1A protein band intensity in Coco-2,compared to the control cells.(3) When microsomes from untreated Caco-2 cell were incubated with N-hydroxy-PhIP ,there was a minor HPLC peak at the expected retention time for N-hydroxy-PhIP-N2-glucuronide.This peak was dramatically increased in the sulforaphane-treated cells,suggesting higher activities of glucuronidation of N-hydroxy-PhlP.(4)The sulforaphane induction of the cells led to a dose-dependent increase in the Nrf2 mRNA levels in Caco-2 treated with 15umol/Land 25umol/L at the concentration (P<0.01) . Cytoplasmic labeling of Nrf2 with no nuclear staining was observed in the non-stimulated cells,whereas an intense nuclear labeling was observed in the sulforaphane-treated cells ,and this finding indicated the induction of nuclear translocation of Nrf2 by sulforaphane.Conclusion:(l)Low dose sulforaphane induced significantly UGTIAn UGT1AK 1A10 mRNA levels.These changes were accompanied by an increase in UGTIAI protein and increase in heterocyclic aromatic amine glucuronidation.(2)It has been established that the induction of the phase II enzyme activity by sulforaphane occurs at the transcriptional level and is regulated by a cuacting element that is present in the promoters of the phase II enzyme genes defined as the antioxidant response element( ARE) ,moreover a member of the basic leucine zipper transcription factor family, Nrf2 (NF-E2-related factor 2), is involved in the activation of ARE. Paper fourExperimental investigation on stimulation apoptosis by sulforaphanein Caco-2 cellBackground: The consumption of cruciferous vegetables has a protective effect on the development of colorectal cancer.The protective effect displayed that the phytochemical sulforaphane is an isothiocyanate found almost exclusively in cruciferous vegetables and can be potent inhibitor of phase I enzymes and inducers of phase II detoxification enzymes.Recently evidence was presented that these phytochemicals produced a marked effect on DNA levels and affected signal transduction pathway ,leading to cell cycle arrest and apoptosis. Sulforaphane , a derivative of isothiocyanates, when inducing UGTIAmRNA expression in our research,can cause morphological changes in Caco-2 treated with more than 35jimol/L sulforaphane.Therefore we had consideration for high-dose sulforaphane may prevent colorectal cancer by stimulating apoptosis.ObjectiverTo investigate on induction apoptosis in Caco-2 by sulforaphane and to elucidate the role of the antineoplastic mechanism in colorectal cancer prevention from morphological changes of apoptotic cell, biochemical variation and apoptotic mechanism and so on .Methods: A human colon cancer cell line Caco-2 was cultured in vitro ,and cells in logarithmic growth phase were selected and examined.(1) The antiproliferative affect of sulforaphane of 10-lOOMmol/L at concentration was examined by means of MTT assay ,and cell survival rate and cell growth inhibition curve were observed and then IC50 calculated.(2) Experimental sulforaphane concentrations were 25u.mol/L> 50umol/L, 75u.mol/L^ 100u.mol/L according MTT assay.(3)To observe colony forming ability by sulforaphane in Caco-2.(4)Morphological variations of apoptotic cell,such as the were observed under invert phase-contrast microscopy, scanning electron microscopy and transmission electron microscopy.(5)Cell cycle analysis and apoptosis rate were evaluated by flow cytometry(FCM) after staining by propidium iodide,and DNA fragmentation was assayed by agraose gel electrophoresis.(6) Apoptosis of Caco-2 was studied by TUNEL ,and apoptosis index were calculated.(7)The mRNA expression of bcl-2,bax gene in Caco-2 were detected with in situ hybridization.(8) Statistical analysis(student t test ,one-way ANOVA and pearson correlation analysis) were carried out using SPSS 11.0 software package.Results:(1) The cell growth was inhibited at various concentrations of sulforaphane(30-100|4,mol/L) in a time-and dose-dependent manner. At the concentration of 50u.mol/L, cell growth suppression rate was dramatically rising and IC5o=50umol/L.(2)Colony forming ability assay showed significant inhibition in Caco-2 treated with 50-100umol/L sulforaphane,and indicating dose-dependent effects.(3) Under the invert phase-contrast microscope,the cell morphology obviously changed while treated with 50-100umol/L concentrations of sulforaphane.Some cells became much more round and brighter, shrinking with the membrane crimpled .Increase vesicles in the cytoplasm and condensed chromatins which attached to the nuclear membrane were observed in treated cells.Many cells fell off the wall of medium and were floating in the culture medium.(4)Under the scanning electron microscope,Caco-2 cells showed typical apoptotic morphological changes after treatment with 75Wnol/L sulforaphane for 36h,characterized by cellular roundness,volume shrinkage and membranous crinkle.The changes of microvillus were very distinct,curled and shortened.Some Caco-2 cells produced apoptotic bodies in the form of bud,and some apoptotic bodies had fallen off from the cellular surface.(5) Under transmission electron microscopy,cell ultrastructural changes were observed as decreased volume,condensed nuclear chromatin and break of membrane...