Inhibitory Effect Of Combined With PI3K Inhibitor And MEK Inhibitor On The Proliferation Of Cisplatin-resistance Ovarian Cancer Cell Line

Objective：To investigate the effect and the possible mechanism ofphosphatidylinositol3kinase (PI3K) inhibitor LY294002combined withmitogen activated protein kinase kinase (MEK)inhibitor AZD6244on theproliferation of cisplatin-resistance ovarian cancer cell line(SKOV3/DDP).Methods：（1）The drug resistance of SKOV3/DDP was determined by MTTassay.（2）MTT assay was used to detecte the alteration in the cellproliferation of SKOV3/DDP cells treated with5,10,20μmol/L LY294002and1,2,4μmol/L AZD6244alone or together; The50%concentration ofinhibition(IC50) and combined index (CI) were calculated.（3）The concentration of the combination is obtained based on theMTT results, and the cells were divided into four groups: the contorl group,the LY294002group(5μmol/L), the AZD6244group(7μmol/L) and thecombination group (LY2940025μmol/L+AZD62447μmol/L); Forty-eight hours later, MTT assay was used to draw the growth curve of cells.(4) Calculating cell doubling time of each group.(5)Colony formation assay was performed to detect colony formationefficiency of each group.（6）Annexin Ⅴ-PE/7-ADD staining combined with flowcytometry(FCM) was used to detect the apoptotic rates and cell cyclestages of each group.（7）Western blotting was employed to analyze the level of AKT,phosphorylated AKT （P-AKT）, ERK1/2, Phosphorylated ERK1/2（P-ERK1/2）, CyclinD1and cleaved Caspase-3protein.Results：（1）IC50of cisplatin to SKOV3/DDP cells was2.3512μg/ml, IC50ofcisplatin to SKOV3cells was0.3035μg/ml, the resistant index of SKOV3/DDP cells was7.7471.（2）Cell growth was inhibited by LY294002andAZD6244alone, theIC50treated with LY294002and AZD6244alone or together forty-eighthours were as follows:(30.69±1.12) μmol/L，(76.26±2.17) μmol/L，（11.75±0.19）μmol/L, The LY294002was5μmol/L and the AZD6244was7μmol/L when they combined, respectively was1/6and1/10of singledrug,CI＜1,the two inhibitors showed synergy effect.（3）The proliferation of the combination group cells was significantlyslower than the LY294002group，the AZD6244group and the control group.（4）The cell doubling time in different groups were as follows:thecontrol group（37.43±0.53）h，the LY294002group（38.95±1.36）h， theAZD6244group（42.15±1.96）h，the combination group（46.92±1.39）h.The cell doubling time of the combination group was significantlyprolonged than single drug group and the control group (P＜0.05).（5）The colony number of the control group, the LY294002group,the AZD6244group, the combination group were as follows:（115±4.51）/103cells，(90.34±4.73）/103cells，（79.67±3.51）/103cells，（62±6.25）/103cells. The colony number was decreased after the combined treatment.(P＜0.01).（6）The apoptotic ratio of the control group, the LY294002group，the AZD6244group, the combination group were as follows:（2.67±0.37）%，（12.08±3.22）%，（20.35±0.97）%，（52.39±3.52）%. FCM demonstratedthat the apoptotic ratio of the combination group was significantly higherthan that of the other group. The G1phase cell proportion were as follows:(40.15±2.70)%,(54.00±1.58)%,(63.84±1.02)%,(77.84±1.26)%, while theS phase cell proportion were:(43.05±4.79)%,(28.47±1.50)%,(23.10±3.25)%,(10.64±0.77)%. In the combination group， the cellssignificantly increased in G1phase and decreasd in S phase(P＜0.05).（7）As western blotting showed, there were no difference in theexpression of AKT and ERK1/2protein among the four groups, in the LY294002treatment group, the level of P-AKT protein decreased and P-ERK1/2protein increased; in the AZD6244treatment group. The level ofP-ERK1/2protein inhibited and the P-AKT increased.;In the combinationgroup, the level of P-AKT, P-ERK1/2and CyclinD1protein weresignificantly inhibited, the cleaved Caspase-3protein was up-regulated(P＜0.05).Conclusion：Combined PI3K inhibitor LY294002and MEK inhibitorAZD6244has synergy effect on inhibition of SKOV3/DDP cell growth byinducing apoptosis and blocking cell cycle. This research provided adifferent theoretical basis for the treatment of cisplatin-resistance ovariancancer.