Inhibitory Effect Of1-Methylhydantoin And5-Fu On Colonic Cancer SW480Cells

Objective:To analysis the inhibit proliferation and induced apoptosis effects of1-Methylhydantoin or in combination with5-fluorouracil in human colonic cancercell line SW480.Methods:To cultivate human colon cancer SW480cells in vitro as the study abject,Theexperiment was divided into four groups: control group（PBS）,1-Methylhydantoin （M）,5-fluorouracil （FU） and1-Methylhydantoin combination with5-fluorouracil （M+FU）.1．MTT assay MTT assay was used to detect inhibition rate①According to literature data and preliminary experiments1-Methylhydantoin wasdivided into eight concentrations (0.5mg·L^-1、5mg·L^-1、10mg·L^-1、20mg·L^-1、50mg·L^-1、100mg·L^-1、200mg·L^-1、500mg·L^-1).1-Methylhydantoin respectively effect onSW480cells after dependent time （24、48、72h）. MTT assay was used to detectproliferation inhibitory effect of1-Methylhydantoin.②Proliferation Inhibition of1-Methylhydantoin and5-fluorouracil to SW480cells: According to the literature data and preliminary experiment,1-Methylhydantoin5-Fu respectively set four same concentration groups, and than compare inhibitioneffect. M (10、20、50、100mg·L^-1),Fu (10、20、50、100mg·L^-1) respectively effecton SW480cells after48h. MTT assay was used to detect proliferation inhibitoryeffect of1-Methylhydantoin and5-fluorouracil.③Proliferation Inhibition of1-Methylhydantoin combination with5-fluorouracil to SW480cells: Combination group was set up four groups: M10mg·L^-1＋Fu10mg·L^-1、M20mg·L^-1＋Fu20mg·L^-1、M50mg·L^-1＋Fu50mg·L^-1、M100mg·L^-1＋Fu100mg·L^-1. Concentration of above groups respectively effect onSW480cells after48h. MTT assay was used to detect proliferation inhibitory effect of5-fluorouracil and5-fluorouracil combination with1-Methylhydantoin. 2. Observe cell morphology by use of microscope:①M100mg·L^-1、Fu100mg·L^-1respectively effect on SW480cells afterdependent time（24h,48h, and72h）, then observe the morphology change of SW480cells with ordinary microscope.②M50mg·L^-1、Fu50mg·L^-1effect on SW480cells after48h, and thenapperceive the morphology change of SW480cells with ordinary microscope afterHE staining.3. FCM detect apoptosis and cell cycle:In order to detect the effect of1-Methylhydantoin to cells apoptosis and cell cycleof SW480.1-Methylhydantoin and5-fluorouracil effect on SW480cells. Each groupof above respectively effect on SW480cells after48h, FCM detect apoptosis and cellcycle①Group1-Methylhydantoin set up two concentrations：1-Methylhydantoin20mg·L^-1、1-Methylhydantoin50mg·L^-1.③Group5-fluorouracil set up one concentration：.5-Fu20mg·L^-1.②Group1-Methylhydantoin5-fluorouracil set up two concentrations：M20mg·L^-1＋Fu20mg·L^-1、M50mg·L^-1＋Fu20mg·L^-1；Results:1. MTT assay results of proliferation inhibitory effect of1-Methylhydantoin inexperimental groups show that:①1-Methylhydantoin could suppress proliferation of SW480cells within therange of experimental concentrations(5～500mg·L^-1), There are statistically significantdifferences contrast between two groups（P＜0.05）.Proliferation inhibition ratedependented on the density of1-Methylhydan-toin and showed a trend of increasing.②1-Methylhydantoin exhibited a highest inhibition ratio on colonic cancer cellline SW480after effecting48hour.③Compared with5-FU(10、20mg·L^-1), there are no significant differences of1-Methylhydantoin suppress proliferation of SW480cells（P＞0.05）; Compared with5-FU(50、100mg·L^-1), there are significant differences of1-Methylhydantoin suppressproliferation of SW480cells（P＜0.05）, The proliferation inhibitory effect of5-FU was stronger than1-Methylhydantoin.④Compared with1-Methylhydantoin and5-FU respectively, With equalconcentrations，there are significant differences of1-Methylhydantoin combinationwith5-FU suppress proliferation of SW480cells（P＜0.05）.⑤Compared with Fu20mg·L^-1，there are no significant differences of1-Methylhydantoin10mg·L^-1combination with5-Fu10mg·L^-1suppress proliferation ofSW480cells （P＞0.05）。There are significant differences of1-Methylhydantoin50mg·L^-1combination with5-FU50mg·L^-1suppress proliferation of SW480cells betterthen Fu100mg·L^-1（P＜0.05）。2. Microscope results of observing the morphology change of different experimentalgroups cells show that:①1-Methylhydantoin and5-Fu with a special concentration (100mg·L^-1)respectively effect on SW480cells after dependent time. The cell volume and nucleusvolume reduced, the number of cell nucleolus and pleomorphism reduced, stainingbecome weak, intercellular junction become loose and partial cells no longer attachingwere observed under microscope.②Observing with microscope after HE staining Compared with control group,the SW480cells morphology changed in experiment group. Such as, cells becomerounded and loose, condensation and margination of cell nuclear chromatin,fragmentation of nuclears and condensation of cytoplasm.3. FCM assay results: Compared with control group,there are significant differencesof apoptosis rate and cell cycle of SW480cells in each treatment group of1-Methylhydantoin、5-Fu（P＜0.05）。Compared with Fu2group,there are significantdifferences of apoptosis rate and cell cycle of SW480cells in group M20mg·L^-1＋Fu20mg·L^-1、M50mg·L^-1＋Fu20mg·L^-1. And cell cycle in S phase gradually increased,cell apoptosis rate gradually ncreasedConclusion:1.1-Methylhydantoin could inhibit proliferation and induce apoptosis of coloncancer SW480cells in vitro.2. In low concentration, proliferation inhibitory to SW480of1-Methylhydantoinequal to5-FU. In low concentration, proliferation inhibitory to SW480of5-FU stronger than5-fluorouracil.3.1-Methylhydantoin and5-fluorouracil have synergistic antitumor effect...