Studies On Antitumor Activity Of Sulindac In Vitro

AIM:To study the effect of sulindac on the colon carcinoma in vitro and to explore its mechanism.METHODS:1.The growth inhibition effect of sulindac was study in different concentrations and different time on Lovo cells by MTT.2.The effect of sulindac induced-morphological change on Lovo cells was observed in different concentrations and different time by inverted phase contrast microscopy.3.The effect of sulindac induced-apoptosis on Lovo cells was observed by transmission electron microscopy,acridine orange flurescence staining method (AO),and flow cytometric analysis.4.The effect of cell cycle of Lovo cells induced by sulindac was observed by flow cytometric analysis.5.The genes differentially expression in Lovo cells induced by sulindac was investigated by cDNA microarray.RESULTS:1.Following sulindac treatment at different concentrations for 24,48,and 72 hours,reduction of cell viability was time-and dose-dependent.The IC₅₀we got in 24,48 and 72 hours were 1.927 mmol/L,0.866mmol/L,0.408 mmol/L respectively.2.Under inverted phase contrast microscopy,we found that the Lovo cells changed their morphology from multiage-type or spindle-type,lessen,more rounder and shrinker,the tight junction of intercellular to vanish.The cells partly fell off and floated in the culture.The longer time and higher concentration of sulindac the more cells floated,but in the control Lovo cells adherenced better, lucency and better photoasty.Under microscopy,characteristic morphology typical for apoptosis was observed at the dose of 0.9mmol/L for 48h including cells shrinkage;vacuoles in cytoplasm,pyknosis,anise chromatin,chromatin margination and complete nuclear membranes were observed.Compared with the blank control,the amount of apoptotic Lovo increased when incubated with the same concentration for the same time observed by AO fluorescence staining.3.The apoptosis was detected by flow cytometric analysis:cells treated with sulindac,the cell apoptotic rate(AR)was higher than that of blank control, and the dose-effect and time-effect relationship were described.Sulindac arrested the cell cycle in G₀/G₁ phase,cells in S and G₂/M phase decreased cells in G₀/G₁ phase increased,and in dose-dependent manner.4.Before and after treated with sulindac(0.9mmol/L)for 48h,the cDNA were extracted and hybridized to the human 17 K cDNA microarray.Among the 17101 genes,1013 genes(1.8%)were differentially expressed,including 434 genes up-regulated(42.84%)and 60 down-regulated(51.76%).The apoptosis associated genes were 178(17.87%),82 of which up-regulated and 96 down-regulated.These differentially expressed genes included apoptosis -related genes,tumor suppression genes,cell receptor and skeleton related genes, immune related genes,signal-transduction-related genes,and development related genes.The genes up-regulated obviously included:GDF15,AREG, ASNS,KLF2,EGR1,IL—11,SESN2,CDC16,MEP1A,S100P,PRO1073, JUNB,SH3GL1,TXNRD1,PSME2,TM7SF2;The genes down-regulated obviously included:CDCA3,CDCA8,KIF20A,BIRC5,C22orf18,ESPL1, NONO,UBE2C,CCNA2,CENPF,PLAU,KIF11,DLEU2,MYBL2,CENPE,HIST1H4F,CNAP1,C10orf3,DLG7,BIRC5,CCNB2,GTSE1,ANLN, TCF19,PPP1R16A,UHRF1,AURKB,CAV1,PLK1,MGC16044,STK6.CONCLUSIONS:The growth of Lovo cells was inhibited obviously by sulindac in vitro,the possible mechanisms may included cell cycle arrested in G0/G1 phase,induced cell apoptosis,regulated some apoptosis -related or cell cycle-related genes.