Changes Of Gene Expression Profiles In Human Hepatocelluar Cancer Cell Line SMMC-7721 And Human Nasopharyngeal Carcinoma Cell Line CNE2 By Telomerase Inhibitors

Telomerase,which is constituted with the RNA and protein components,is a special DNA polymerase with reverse transcriptase activity.The RNA component acts as the template and synthesizes the telomeric DNA repeat sequences TTAGG to the terminal end of a chromosome.It has been confirmed that telomerase plays a considerable role in tumorigenesis and tumor development.Telomerase activity has been found in nearly 90%of all human cancers,but not in most normal cells and benign tumor cells.Presently,telomerase has been regarded as a new tumor marker and explored as a promising tool in cancer diagnosis and therapy.Many scholars dedicate to develop telomerase inhibitors of various mechanisms of action and expect that they can work as safe,effective antitumor drugs.However, the studies and researches on telomerase inhibitors have showed that there are multi molecule targets for telomerase inhibitors of different mechanisms in various kinds of cells or even in the same kind of cell.Therefore,the search and identification of the common target sites for different mechanism telomerase inhibitors, especially the common key controlling fators impacting telomerase activity,will help to illustrate and understand the mechanisms of telomerase inhibitors better.Moreover,according to the study, theory evidences for clinical applications of telomerase inhibitors and for developping new types of telomerase inhibitors are provided.Objection:Analysis the changes of gene and protein expression profiles in human hepatocelluar cancer cell line SMMC-7721 and human nasopharyngeal carcinoma cell line CNE2 by different telomerase inhibitors,and discuss the potential target sites of telomerase inhibitors.Methods:1.MTT was used to observe the proliferation of carcinoma cells and the cytotoxicity of the three telomerase inhibitors:EGCG,the antisense oligodexoynuclectide(ASODN),and the sense oligodexoynuclectide(SODN),and the suitable concentrations of various telomerase inhibitors for SMMC-7721 and CNE2 cells was defined.2.Cultured cells were harvested at log phase.SMMC-7721 and CNE2 cells were treated by the telomerase inhibitors(EGCG,ASODN and SODN) with the optimal concentrations.After treated,the total mRNA in all groups was extracted and the gene expression was detected using Affymetrix U133A 2.0 array.Genes changed in common were screened.3.SMMC-7721 and CNE2 cells were treated by eight different telomerase inhibitors:EGCG,ASODN,SODN,ADM,ATRA,AZT,HAODN and HSODN.After treated,the proteins of cell in all groups were extracted.SELDI technology was used to detect the changes of protein expression,and proteins changed in common were screened.Results:1.According to MTT,for both the SMMC-7721 cells and CNE2 cells,the optimal drug concentrations within 48 hours were: EGCG 125.0 mg/L,ASODN 6.0μmol/L,SODN 6.0μmol/L.2.The changes of gene expression profiles in SMMC-7721 cells and CNE2 cells by telomerase inhibitors are in varied degrees.(1)For SMMC-7721 cells,after treated with EGCG,196 genes were differentially expressed,in which 132 genes were up-regulated and other 64 genes were down-regulated;treated with ASODN,the numbers of changed genes,up-regulated and down-regulated genes were 2025,897 and 1128;and numbers for SODN were 2337,1254 and 1083.In 14500 genes,the expression of 14%genes were changed by both ASODN and SODN,when 2%by EGCG.(2)For CNE2 cells,after treated with EGCG,3193 genes were differentially expressed,in which 1217 genes were up-regulated and other 1976 genes were down-regulated;treated with ASODN,the numbers of changed genes,up-regulated and down-regulated genes were 262,158 and 104;and numbers for SODN were 151,34 and 117.In 14500 genes, the expression of 22%genes were changed by EGCG,when less than 2% by both ASODN and SODN.3.Collective changes of gene expression profiles in SMMC- 7721 and CNE2 cells treated by EGCG,ASODN and SODN were as follows:(1)10 up-regulated genes in both SMMC- 7721 and CNE2 cells after treated with EGCG were:BTG1,TXNIP,SGK,LDLR,TRIB1,PER2,HIST1H2AG, H1F0,TFPI,HSPBAP1;8 down-regulated genes were:ID1,ID2,ID3,FABP3, AKR1B10,PTGES,HSPH1,ANGPTL4.(2)11 up-regulated genes in both SMMC- 7721 and CNE2 cells after treated with ASODN were:TIMP3,CYP51A1,NT5E,SPRY2,FGF2,AREG, SC4MOL,DDIT3,ETV4,RASA4,PHLDA1;9 down-regulated genes were:DCN, AKR1B10,MR1,MUC1,UGT1A10,AKR1C3,LXN,TMEM45A,UGT1A8. (3)2 up-regulated genes in both SMMC- 7721 and CNE2 cells after treated with ASODN were:SERPINE1,KRTHA4;7 down-regulated genes were: KIAA0152,ANP32A,NPTX1,AP1S1,HMGCS1,HIST1H4C,SC4MOL.(4)In SMMC-7721 cells,12 genes were up-regulated by all the three telomerase inhibitors respectively:BTG1,WSB1,MXI1,CCNG2,BCL6, WASL,SCAMP1,H1F0,RALGDS,FOS,TFPI,KLHL24;4 genes were down-regulated:DI02,AKR1B10,ID3,AKR1C1.(5)In CNE2 cells,there was only one up-regulated gene KRTHA4 by all the three telomerase inhibitors respectively;Only one gene CA5B was up-regulated.(6)The common changed genes were closely related to a range of biological functions including signal transduction,cell cycle, transcription regulation,apoptosis,energy metabolism and so on.4.The changes of protein expression profiles in SMMC-7721 cells and CNE2 cells by the eight telomerase inhibitors:ADM,ATRA,AZT, ASODN,SODN,EGCG,HASODN and HNODN.(1)For SMMC-7721 cells,after treated with the eight telomerase inhibitors,there were 28,20,33,12,24,18,10,23proteins down-regulated and 6,20,7,41,13,25,42,20 proteins up-regulated respectively.(2)For CNE2 cells,after treated with the eight telomerase inhibitors,there were 14,26,27,23,31,42,26,30 proteins down-regulated and 22,11,18,18,7,7,13,8 proteins up-regulated respectively.5.Collective changes of protein expression in SMMC- 7721 and CNE2 cells treated by ADM,ATRA,AZT,ASODN,SODN,EGCG,HASODN and HNODN were as follows: (1)For SMMC-7721 cells,6 proteins(2477.89 Da,3723.96 Da, 3898.37 Da,4067.51Da,4127.75 Da and 4216.32 Da)were down-regulated by all the eight telomerase inhibitors respectively;2 proteins (2205.47Da and 6647.26 Da)were up-regulated.(2)For CNE2 cells,only one protein(2657.14 Da)was down-regulated by all the eight telomerase inhibitors respectively;no protein was up-regulated by all the eight telomerase inhibitorsConclusions:1.In SMMC-7721 cells or CNE2 cells respectively,there were collective changes of gene expression profiles treated by EGCG,ASODN and SODN.There were also collective changes of protein expression in SMMC-7721 or CNE2 cells respectively after treated by ADM,ATRA,AZT,ASODN,SODN,EGCG,HASODN and HNODN.The genes and proteins changed in common were probably the key points which involved in inhibition of telomerase activity.Maybe they were the common target sites effected by different telomerase inhibitors.2.There were no genes changed in common by all the three telomerase inhibitors(EGCG,ASODN and SODN)in both SMMC-7721 cells and CNE2 cells,so were the proteins changed in common by all the eight telomerase inhibitors(ADM,ATRA,AZT,ASODN,SODN,EGCG,HASODN and HNODN).Possiblely telomerase inhibitors had different target sites and works in diverse mechanisms in differnet cancer cells.