The Study On Anti-B16 Mouse Melanoma Cells Effect And Mechanism Administrated By Artesunate With Paclitaxel

Objective1.This study is the effect on the B16 mouse melanoma cells administrated by artesunate(ATS),dihydroartemisinin(DHA),Paclitaxel(Taxol)and ATS with Taxol.Experimental method supports the single and combination drug anti-mouse melanoma B16 cells proliferation in vitro.2.The study is about the mechanism of ATS anti-B16 mouse melanoma cells.It is observed the migration ability,the necrosis/apoptosis and the cell cycle.And monitored the change of genes and protein by real time-Polymerase Chain Reaction(PCR)and Western Blot.Methods1.Detected the half inhibitory concentration(IC50)of ATS,DHA and Taxol inhibited the proliferation of B16 mouse melanoma cells for 24 h and 48 h by applying the CCK-8 method,and the combination of AT(1:1),AT(3:2)and AT(7:3)for 48 h.Then analyzed the combination by the effect Median-effect Equation,the Unified Theory of the Mass-Action Law analysis and the cell morphological observation.2.With scratch experiment to observe the ATS(0.25,1.00 ?M),Taxol(0.25,1.00 ?M)and the combination ATSO.175 ?M+TaxolO.075 ?M ATSO.7 ?M+Taxol0.3?M)impact on the B16 mouse melanoma cells migration ability,and recorded it by photos.3.With Flow Cytometry(FCM)to detect ATS(0.25,1.00,5.00 ?M)on the B16 mouse melanoma cells 24 h,the experimental results showed the affect of the cells apoptosis/necrotic and cell cycle.4.With Real-time quantitative PCR to monitor the change of genes on ATS(0.25,1.00,5.00 ?M)effected on the B16 mouse melanoma cells 24 h.The migration genes such as MMP2/9;the apoptosis/necrosis genes such as RIP1/3,Fas/FasL,Caspase-3/8;the DNA replication checkpoint genes such as ATR,Chkl,Cdc25C,Cdk1,CyclinB1,cdk1;and the DNA damage checkpoint genes such as ATM,Chk2,p53,p21,Cdc25A,Weel,CyclinDl/E1.5.With Western Blot to defect ATS(0.25,5 microns)effected on the B16 mouse melanoma cells after 24 h,We detected the CyclinB1,CylinDl,Weel and Cdc25C protein expression changes.Results1.The IC50 value of ATS,DHA and Taxol at 24h were 126.56,86.09,3.27?M;and at 48h were 1.36,15.11,0.97?M.And it showed good synergistic effect of the ATS combined Taxol,and.-its three proportion is AT.(1:1),AT(3:2)and AT(7:3)as the combination index(CI)value<1.2.It could be clearly observe that there was no difference between low concentration of ATS(0.25uM)and the blank control group,when the concentration increased to ATS(1uM),it had the ability to inhibit the migration of B16 mouse melanoma cells.For the Taxol group,its effect on the migration of B16 mouse melanoma cells was very obvious,even the low concentration of Taxol(0.1uM)had a very strong ability to inhibit the migration of cells.And the combination group,we got that both the ATS0.175uM+Taxol0.075uM group and the ATS0.7uM+Taxol0.3uM group showed a very strong ability to inhibit B16 melanoma cells in migration.3.Compared with the blank group,ATS(0.25,1,5 ?M)was not effect obviously on apoptosis/necrosis(P>0.05).And the apoptosis cells were not obvious,but the necrosis cells had significant difference(P<0.001),when the concentration of artesunate is greater than 1uM.4.Compared with the blank group 0 ? M),ATS 0.25,1,5 ? M)could obviously block the cell cycle.As the proportion of G0/G1 phase and G2/M phase cells increased significantly,and S phase cells decreased significantly,the difference was statistically significant(P<0.001).With the increase of the proportion of ATS,the effect is stronger.5.For monitor the ability of cells,the expression of MMP-9 mRNA decline was not obvious,but MMP-2 was significantly decreased.Not replicate DNA checkpoint showed that ATR and Chkl/2 mRNA expression increased obviously,but Cdc25C,CyclinBl and cdkl mRNA decreased.And the DNA damage checkpoint showed that ATM,p53,p21 and Weel mRNA expression increased obviously,but Cdc25A,CyclinE1/D1 mRNA decreased.6.The CylinD1,CylinB1 and Cdc25C protein expression dropped obviously with the increase of concentration of ATS(0.25?5 ?M),but Weel protein expression increased.Compared with blank control group,the ATS(5 ?M)group had a significant statistical difference(P<0.01).Conlusions1.ATS,DHA and Taxol anti-B16 mouse melanoma cells,with the concentration and the time increase,the proliferation inhibition significantly increase;And ATS combined Taxol have a very well synergy effect to inhibit the proliferation of the B16 mouse melanoma cells.2.ATS can effectively inhibit the migration ability of B16 mouse melanoma cells,it may be associated with decrease the expression of MMP-2.3.ATS can induce B16 mouse melanoma cells apoptosis.It possibly through the Fas/FasL,or the TNF-? induced apoptosis signals may be passed by RIP1 kinase compounds FADD and Caspase-8.4.ATS can effect on B16 mouse melanoma cells to the cell cycle arrest in G0/G1 phase and G2/M phase.For the non-replicate DNA testing point,it possibly by activating the ATR and downstream Chkl,decrease Cdc25C expression,which makes the compound of CyclinB1-Cdkl expression decrease,and blocks the cell cycle in G2/M phase.For DNA damage checkpoint,it possibly by activating ATM/ATR,increase the expression of p53 and Chkl/2.Then p53 activates the downstream p21.Then Chkl/2 also makes Cdc25A expression decrease,which makes the compound of CyclinE1-Cdk2 and CyclinD1-Cdk4/6 expression decrease,and block the cell cycle in G0/G1 phase.Also it possibly by activating ATM/ATR,and the downstream Cdc25C would be decreased,but the Weel.Which also would make the compound of CyclinB1-Cdkl expression decrease,and blocks the cell cycle in G2/M phase.