Cloning And Identification Of Differentially Expressed Genes Related To Human Colon Cancer Cell Growth Inhibition Induced By Diallyl Disulfide

Objective: This study was designed to explore the antiproliferative ability of diallyl disulfide on human colon cancer HT-29 cells, and its related molecular mechanisms.Methods: Cell morphology was observed by inversion microscope and optics microscope.HT-29 cells growth inhibition were measured by cell counting and MTT assay. Phase distribution of cell cycle was analyzed by flow cytometry (FCM). Immunocytochemical staining and morphometric quantitative analysis detected the expression of p21WAF1, c-Myc,Cyclin E and p53 .Results: As exposed to inversion microscope and optics microscope, when 60, 90,120 μ mol/L DADS was added to culture medium 24 h after cell seeding this agent was found to reduce the number of adherent cells in the culture flask up to day 3 of cell culture when compared with cells cultured in the presence of vehicle alone (i.e. DMSO), which by itself has no effect on cell growth . Morphometric quantitative analysis showed that the morphological changes of HT-29 cells that still adhere the flask were not found , as may indicate that DADS can't induce the differentiation of HT-29 cellsMTT assay showed that apparently growth inhibition in HT-29 cell line treated with DADS could be seen and exhibited a dose-dependent model, and at beyond concentration of 60 μ mol/L, DADS displayed also a time-dependent model. Adding 60, 120, 240 μ mol/L DADS for 24 h suppressed HT-29 cells growth by 23.1%,45.6%, 68.3% (P<0.05), and 28.4%, 48.2%, 72.5% (p<0.05) for 48 h.Cell counting showed that average doubling time retarded from 22.58 h in untreated HT-29 cells to 31.2 h in 60μmol·L - 1 , and to 81.2 h. in 120 μmol·L - 1Flow cytometry analysis revealed that treating HT-29 cells with 30, 60μmol/L DADS accumulated cells in the Gl phase, however, 90, caused G2/M arrest. The proportion of cells in the Gl phase after treatment with 60μmol/L DADS for 24 h was 45.2 % , almost 2-foIds that occuring in untreated cells ( 27.6 %) . The proportion of cells in the G2/M phase after treatment with 120 μ mol/L DADS for 24 h was 61.0%, about three times that occurring in untreated cells (23.6%).Immunocytochemical stain and morphometric quantitative analysis indicated that 60μmol/L DADS was associated with an increased amount of p21WAF1 and a decreased amount of c-Myc, Cyclin E protein, and 120μmol/L, DADS exerted the same effect in the expression of p21WAF1, c-Myc on the HT-29 cells. However DADS didn't influence the expression of mutational p53 protein.Conclusion:1. DADS is an effective inhibitor of the growth of human colon cancer HT-29 cells., as may be related to cell cycle arrest.2. Low doses DADS blocked HT-29 cells in G2 may be related to enhance the expression of p21 WAF1 and down-regulation of c-Myc, Cyclin E.3. high doses DADS induce HT-29 cells G2 /M arrest may be related to enhance theexpression of p21WAF1 and down-regulation of c-Myc.Part Ⅱ: Isolating and cloning the differentially expressed genes on the colon cancer cells growth inhibition induced by DADSObjective: This study was designed to isolate and clone the differentially expressed genes related to the antiproliferative ability of diallyl disulfide on human colon cancer HT-29 cells.Methods: Differentially expressed cDNA species induced by DADS on humancolon cancer HT-29 cell line were determined by using suppression subtractive hybridization (SSH).Then these cDNA species were directly insected into T-A cloning vector to set up the forward and reverse subtractive libraries. Amplification of the libraries was carried out with transformation of JM109.100 positive clones were sequenced and analyzed for homology in the Genbank database with BLAST. Ten genes chosen randomly from the two libraries were analyzed by using semi-quantitative reverse transcription-polymerase chain reaction. Results: The RNA abstracted : Total RNA abstracted by TRLzol reagent was determined by agrose gel electrophoresis and three bands ( 28S RNA , 18S RNA and 5S RNA ) were observed.The quality of mRNA was also good, as observed by electrophoresis. rRNA and protein were not obviously found .The experiment of SSH : The subtracted products and unsubtracted products were totally different. By agrose gel electrophoresis, the former appeared clear ban unsubtracted ds; the latter, smear .This indicated that the sequences shared by two groups of cells Were subtracted, only the sequences existed in the tester can be amplified.The subtraction efficient of SSH experiment: Used specific primers of G3PDH gene ( a house keeping gene ) as PCR primers and subtracted products as template , no product can be seen even cycled 30 times . However, when used unsubtracted products as template , obvious products of G3PDH gene can be seen even only cycled 15 times . As a result, it can be said that the subtraction efficient is very high .The experiment of reverse SSH: Contrary to above experiment, used cDNA came from induced colon cancer cells as driver and cDNA from control cancer cells as tester , a new cycle of SSH was performed . By this way , the differentially expressed genes in colon cancer cells were obtained .After T/A clone , the linked products were transferred into JM109.The positive clones ( white clones ) were amplified in LB liquid medium .The amplificd library contained morethan 500 positive bacteria clones . Random analysis of 100 clones with enzyme restriction and PCR methods showed that 73 positive clones contained 100 to 1300 bp inserts.Sequencing results showed that there were 68 clones were identified , another 5 clones could not be identified because without positive results or some of bases were not clear . After comparing with public database ( GenBank / EMBL ) and Expressed Sequence Tag ( EST ), we found that there were 38 known genes and a nove EST in the two libaries. Data showed that those known genes represent a variety groups on the basis of their cellular functions and have 95-100 % identities in sequences. We classified those known genes into four groups, one group of the genes was cell cycle and apoptosis -associated, such as P21, MRPS27,MM1, LGALS8 in the forward substract-library , and PABPC1, YWHAE, RRM1 in the reverse substract-library. The second group of genes was functionally associated with metabolism and antioxidant enzymes, including SOD1, ARPC3, MTO1, citrate synthase, DNCL1, HNRPAB, FTH1, SPINT2, UBA52 in the forward substract-library, and TRIP13, TRA1 in the reverse substract-library. In the category of signal ransduction-related molecules, we found that molecules such as TACSTD2, S100A6, PSMD12, GNB1, GPCR175, NSFLIC in the forward substract library, and ATP1A1, ATP6AP1, FLJ25150 fis, RAC1 in the reverse substract-library.The fourth groups including some unknown genes which function were not identified and a novel EST,they were Ribosomal protein S3, DPCD, LOC144501, MGC41816, RPL35A, HTDP1 ( novel EST) in the forward substract-library, and CLCP1, TMED5, KIAA0056 protein, RPS6, Heat shock protein 8 in the reverse subtract-library. Many genes involved in the antiproliferation of DADS on the HT-29 cells.were described for the first time, and further, some genes unreported may be related to the DADS antitumorigenic activity. Since these genes belong to different physiological systems, DADS seems to have widespread functions.semi-RT-PCR: Using semi-quantitative reverse transcription-polymerase chain reaction, ten genes chosen randomly from the two libraries were analyzed in the untreated and DADS-treated(120 μM) HT-29 cells, the results showed a consistent expression pattern with that of the SSH analysis. Conclusion: 1. The differently expression genes before and after induced by DADS were...