The Study On The Effect Of Proliferation Inhibition Of Human Lung Adenocarcinoma A549Cells And The Effect On MMP-2by Cinobufacini Combined With DDP

Objectives:This experiment through using different concentration of cinobutacini and thechemotherapy drug cisplatin to observe the effects on inhibition of proliferation, cellcycle and apoptosis of lung adenocarcinoma A549cells while treated by a single drugand two drugs combination at different concentration as well as administration time, andthrough using immunohisto chemical method to study the effects of three different planson MMP-2activeness expression, to discuss the possible anti-cancer mechanisms ofcinobutacini, contrasted the proliferation of the single drug and the combinations at thesame time. And then provided experiment theory basis for adopting cinobutacinicombined cisplatin in clinic.Methods:1.Cultured lung adenocarcinoma A549cells in vitro to logarithmic growth phase.2.Through MTT testing the cell growth inhibition rate of lung adenocarcinomaA549cells in different concentrations of cinobutacini (0.1、0.2、0.4ug/ml),differentconcentrations of cisplatin（0.25ug/ml、0.5ug/ml、1.0ug/ml）and combined the two drugsafter intervening24h,48h and72h.3.Using FCM to detect the changes of cell cycle and apoptosis rate of lungadenocarcinoma A549cells in single and combined groups of the cinobutacini of0.4μg/ml and the cisplatin of0.5ug/ml after intervening48h, and observingmorphological changes of these cells through inverted phase contrast microscope.4.Detecting the effects of different groups drugs acting on the protein levels ofMMP-2through immunohistochemical method. Results:1.Inhibition of proliferation of lung adenocarcinoma A549cells affected bycinobutacini and cisplatinBoth cinobutacini and cisplatin have the effect of inhibiting the proliferation of lungadenocarcinoma A549cells, which showed a time-and concentration-dependence, that isto say, the longer time period and higher drug concentration act on cells, the morestronger inhibition effect.2.Morphological changes of these cells after given different concentration ofcinobutacini and cisplatinWhen we gave small concentration cinobutacini and cisplatin, the cells died byshowing apoptosis mostly, their structure transformed from the original spindle shape toround shape, cell adhesion is poor. Increasing the concentration, we were able to observecell vacuoles and a large amount of round-shaped cell debrises by inverted phase contrastmicroscope, showing morphological of cell necrosis.3.Inhibition of proliferation of lung adenocarcinoma A549cells in drug combinationgroupCombining0.5ug/ml cisplatin with0.1ug/ml、0.2ug/ml and0.4ug/ml cinobutacinirespectively, the inhibition of human lung adenocarcinoma A549cells was higher thanmonotherapy groups. The higher drug concentration, the higher inhibition. Especiallyafter48hours, the inhibition is more obvious.4. Effect of single drug use and combination drugs on cell cycle and apoptosisAfter intervening48h,0.5ug/ml cisplatin and0.4ug/ml cinobutacini blocked cells atS phase （p<0.01); compared with single drug groups and the control group,0.5ug/mlcisplatin combined with0.4ug/ml cinobutacini, cells at S phase was increased,whichshowed blocked cells at S phase. The combined group induced apoptosis significantlygreater than the single drug group, compared with the control group having significantdifferences (p<0.01).5.The effects of cinobutacini, cisplatin and the combination of the two drugs on the expression of MMP-2Both cinobutacini and cisplatin could inhibit the activation of the expression ofMMP-2and we could not find difference between them,so there was no statisiticalsignificance (p<0.0083).Combination found that they could obviously inhibit theexpression of MMP-2,comparing with the single group, the effects were significantlybetter(p<0.0083).Conclusions:1.Both cinobutacini and cisplatin have the ability to block cell cycle progressionwhile display time-and concentration-dependence in some time.2.Comparing with the single drug group, the combination group had moresignificantly proliferation inhibition.3.Both cinobutacini and cisplatin have the ability to block cels at S phase, and thecombination group cell apoptosis rate was significantly higher than the single druggroup.4.The anti-cancer processof the cinobutacini associated with the expression ofMMP-2,Both the single drug group and the combination group could reduce theexpression of MMP-2, and comparing with the single drug group, the effects of thecombination group were significantly enhanced.