Effect Of Different Time Of Trageted Drugs Associate With Cisplatin On The Induction Of A549Apoptosis

Objective:In this study,the lung adenocarcinoma A549cell is the object ofstudy.Choose the nimotuzumab and trastuzumab which are monoclonalantibody against EGFR and HER-2to combine with cisplatein in differenttime. Through vitro experiments,to observe partly the effect of A549cellprolifation inhibition when nimotuzuab and trastuzumab combined withcisplatin monotherapy in different time. This study aims to provideexperimental basis for targeted selection and combination drugs duringclinical treatment.Methods:（1）By MTT way to partly test the OD value of combiningnimotuzumab and trastuzumab in different time and the two combiningtargeted drugs different time with cisplatin，then calculate the inhibitory rate ofcell growth and get the half inhibitory rate (IC50) of each group’ cisplatin.Draw cell growth inhibition rate-drug concentration curve.（2）Check thedistribution of A549cell cycle in each group by FCM Flow Cytometry.（3）By Western Blot to check the expression levels of A549cells’cyclin D1ineach group.Results:Experiment ONE,Group A (Different concentrations of monotherapycisplatin), Group B((hR3DDP sequential mode), C group (hR3and DDPsimultaneous mode), D group (blank control group).MTT show A, B, C three groups have significantly prolifation inhibitioneffect on the A549cells,and positively correlate with the concentration andtime.The inhibition ration of Growp B was more prominent than Group A,andthe inhibition ratio after72hours reaches the highest ratio (96.48±0.76). But the prolifation inhibition ratio decreased in Group C than Group A. The IC50ofcisplatin in three groups were as follows:1.58±0.02,0.74±0.03,2.42±0.06ug/ml.There was a significant statistically difference compared this threegroups(P<0.01). The cell growth inhibition rate-the drug concentration curveof Group B shifted right when compared with Group A. But the Cell growthinhibition rate-the drug concentration curve of Group C shifted left whencompared with Group A.Flow Cytometry shows that the percentage of A549cells in G0-G1phrase were66.40±2.31%,72.23±2.07%,62.28±1.68%,58.25±1.07%respectively after the A.B.C.D groups worked in48h. The percetage of cells inphase G0-G1of Group B was significantly increased when compared withGroup A,but the percentage of cells in phase G0-G1of Group C wasdecreased than Group A(P<0.05).Western Blot shows that the protein Cyclin D1expression level(use thegrey value) of the Group A.B.Cand D were as follows:0.31±0.34、0.24±0.19、0.40±0.28、0.56±0.20.The cyclin D1expression of each group havereduce in different level when compare with the Group D. The expressionof Cyclin D1protein in Group B decreases obviously when comparedwith Group A (P<0.05).The expression of Cyclin D1protein in GroupC is higher than Group A (P<0.05).Experiment Two, Group A (different concentrations monotherapy DDP),Group B(Herceptin DDP sequential mode), C group (Herceptin and DDPsimultaneous mode)MTT shows A, B, C three groups have significantly prolifation inhibitioneffect on the A549cells,and positively correlate with the concentration andtime. There is no significant statistically between Group A and Group B（P>0.05）.There are statistically difference between the Group A、C andGroup B、C(P<0.05）. The inhibition rate of Group C is lower than the GroupA and Group B. Compare the IC50of cisplatin in three different time points ofeach group：There was no significant statistically between Group A and GroupB（P>0.05）.There were difference between the statistics of Group A、C and Group B、C(P<0.05).The IC50of Group C was higher than Group A andGroup B.Experiment Three, Group A(monotherapy DDP mode),Group B(hR3sequential with DDP mode).Group C(Herceptin sequential with DDPmode),Group D(hR3combined Herceptin sequential with DDP mode), GroupeE(hR3combined Herceptin synchronous with DDP mode), Group F(blankcontrol group).MTT shows that five groups drug have significantly prolifation inhibitioneffect on the A549cells,and positively correlate with the concentration andtime.The proliferation inhibition ratio of five group of A549cells after72htreatment were as follows:56.97±1.42,82.17±1.62,60.13±2.56,90.97±1.32,60.01±1.61.The differences have statistically significant(P<0.05). Theinhibition ratio of Group D is the highest,the second is Group B,but there is noclearly difference between Group A,Group C and Group E(p>0.05).Flow Cytometry shows that the percentage of A549cells in G0-G1phrase were66.40±2.31%、72.23±2.07%、66.26±2.06%、75.52±0.57%、66.71±1.24%、58.25±1.07%respectively after the A.B.C.D.E.F six groupswork in48h.The percentage of cells in phase G0-G1，Group A,Group B,GroupC,Group D and Group E were significantly increased compare to blank controlgroup(P<0.05). Group D is the highest,and there is no statistically significantwhen compared the cells of Group A、C and E in G0-G1phase (P>0.05).Western Blot showed that the protein Cyclin D1expression level(use thegrey value) of the GroupA.B.C.D.E and F were as follows:0.31±0.34、0.24±0.19、0.33±0.10、0.12±0.14、0.32±0.11、0.56±0.20.The cyclin D1expression of each group have reduced in different level when compared withthe Group F. The expression of Cyclin D1protein in Group D decreasedobviously (P<0.05).Conclusion：The Inhibition rate of nimotuzumab sequential cisplatin is increasedwhen compared with cisplatin monotherapy. This illustrates that nimotuzumab can enhance the anti-tumor effect of cisplatin, while when use them at thesame time will play the role of antagonism.Trastuzumab sequential cisplatin compares to cisplatin monotherapyfinds the inhibit rate have no change. But the inhibition rate is lower than thecisplatin monotherapy when trastuzumab compared with cisplatin.Thisillustrates that trastuzumab can not enhance the anti-tumor effect ofcisplatin, while when use them at the same time will play the role ofantagonism. Sequential with two kinds of targeted drug cisplatin can improvethe human lung adenocarcinoma A549cell proliferation inhibition, byreducing the levels of CyclinD1,to make the A549cells be inhibited inG0/G1phase.This provides some theoretical basis for the clinical where firstuse the target drug and then combine chemotherapy drugs with target drug.