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Effects and Mechanism of Ardipusilloside-Ⅰon Cell Invasion and Migration Ability of Glioma Cells

Author: WangLin
Tutor: LiXiaoBing
School: Luzhou Medical College
Course: Pharmacology
Keywords: Ardipusilloside-I Glioma cell Migration Invasion Matrixmetalloproteinase
CLC: R273
Type: Master's thesis
Year: 2013
Downloads: 7
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Abstract


Objective: To investigate the the influence of Ardipusilloside-I, a triterpenoidsaponin isolated from Ardisia pusilla A. DC on invasion and migration ofglioma cell line U87cells and evaluate its mechanism. Methods: U87cellswere cultured without bacteria in vitro, the effect of different concentrationsof Ardipusilloside-I（0、2、4、8、16、32μM） on U87cells viability weremeasured by3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay, then observed the effect on different time（24、48、72h）.Themodel of transwell chamber which with50mg L-1matrigel-coated for imitatingthe extracellular matrix in vivo was used to investigate the effect of invasioncapabilities of U87cells with different concentrations of Ardipusilloside-I（1、3、5μM）after24h. The migration capabilities of U87cells were assessed usingwound healing assay after dealing with different concentrations ofArdipusilloside-I（1、3、5μM）and different time（0、6、24h）in6-well plates.Matrix-metalloproteinase (MMP) was determined after using differentconcentrations of Ardipusilloside-I （1、3、5μM）on U87cells by gelatinzymography on8%polyacrylamide gel containing1g L-1gelatin. Throughcorresponding the antibody and antigen, detected the positive reaction ofMMPs in U87cells with antibody at different concentrations Ardipusilloside-I（1、3、5μM）in immunochemical assay. Results: MTT assay showed Ardipusilloside-I inhibited the proliferation of glioma U87cells, and thetendency in dose-dependent and time-dependent manner, the IC50valuescalculated from the dose effect curve were8.02±0.16μM、6.63±0.10μM、5.41±0.05μM for Ardipusilloside-I at24h,48h and72h incubationrespectively. Then choose the effective time of Ardipusilloside-I on cells was24h and the drug concentrations were1、3、5μM from this assay as standard forother experiments. Compared with control group, the migration and invasion ofU87cells treated with Ardipusilloside-I were decreased significantly, theinhibition ratio of invade with3and5μM Ardipusilloside-I-treated U87cellsboth more than45%, but not obviously in low concentration. At wound healingassay the gap distance was significantly reduced in U87cells byArdipusilloside-I at high concentration and long time. At24h, the migrationratio of control group was55.44%, and the migration ratio of Ardipusilloside-I-treated U87cells were50.02%at1μM and25.97%at3μM and12.99%at5μM.And the MMP-2activity also decreased through gelatin zymographyassay compared with control group, the inhibition ratio was not obviously atlow concentration after24h, but the Ardipusilloside-I can reduce the secretionof active MMP-2from U87cells, and the inhibition ratio were73.22%and61.53%respectively at the dosage of3μM and5μM of the Ardipusilloside-I.Through immunocyto chemical analysis of MMP-2proteins expression in U87cells, the DAB chromogenic showed color of control group and1μM groupwere dark claybank（+++）, the color of3μM group was claybank and the color of5μM group was sundown, and with the higher of the concentration ofArdipusilloside-I, the lower value of IOD, so it indicated the rate of positiveactivity of MMP-2was more lower with the increase of the concentration ofArdipusilloside-I, the Conclusion: The results suggest that Ardipusilloside-Imay inhibit the migration and invasion of glioma U87cells by suppressing thematrix metalloproteinase-2activity.

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