LY294002Increased The Sensitivity To As₂O₃of Acute Myeloid Leukemia Cells Co-cultured With HS-5

Objective:To investigate the sensitivity to arsenic trioxide (As2O3) of acutemyeloid leukemia(AML) cells after co-cultured with bone marrow stromal cell HS-5increased by LY294002.Methods:Human AML cell lines (NB4, HL60, or U937cells) was co-cultured withHS-5to mimic the growth of AML cells in vivo and CD45+magnetic bead was usedto sort out the AML cells which were directly adhered to stromal cell.MTT and trypanblue staining assay were performed to determine the inhibitory effect of drugs onAML cells.Annexin-V-FITC/PIdouble staining flow cytometry was utilized to detect theapoptosis cells.Cell cycle distribution was measured by flow cytometry in AMLcells.Western blot was executed to detect the protein expression level of p-AktSer47,p-AktThr308, p-PDK1and p-PTEN.Results:The proliferation inhibition rate of AML cells was inhibited,which AMLcells treated with As2O3for72hours.And the suppression of AML cells wasdependent on the concentration and incubated time of As2O3.The highest sensitivity toAs2O3on NB4cells was observed in the three AML cells.The proliferation inhibitionrate of NB4, HL60, or U937cells co-cultured with HS-5to As2O3was decreasedcompared with the three AML cells cultured single,(15.86±3.01)%VS (62.92±2.74)%,(14.96±1.40)%VS (48.54±4.54)%and (19.19±2.39)%VS (52.53±2.99)%respectively.However,LY294002combined with As2O3increased the proliferationinhibition rate of NB4, HL60, or U937cells co-cultured with HS-5compared withAs2O3only,(33.95±4.35)%VS (15.86±3.01)%,(34.77±2.07)%VS (14.96±1.40)%and(38.09±2.24)%VS (19.19±2.39)%respectively.After co-cultured with HS-5,the proportion of apoptotic cells in NB4, HL60, orU937cells induced by As2O3was significantly decreased compared with culturedsingle,(13.60±0.85)%VS (23.44±2.37)%,(9.84±1.99)%VS (17.97±1.58)%and (7.28±1.78)%VS (17.76±2.13)%respectively.The proportion of G0/G1phase in NB4, HL60,or U937cells co-cultured with HS-5for one day was significantly increasedcompared with the three AML cells cultured single,(52.53±2.39)%VS (32.47±2.8)%,(47.02±3.42)%VS (30.31±2.07)%and (63.02±2.46)%VS (41.46±2.58)%respectively,while the proportion of S phase was significantly decreased,(36.87± 3.28)%VS (60.35±4.88)%,(45.45±4.33)%VS (62.33±2.27)%and (28.53±2.44)%VS(51.56±3.69)%respectively.Akt and PDK1phosphorylated protein of AML cells was down-regulated treatedwith LY294002for2to12hours,while PTEN phosphorylated protein wasup-regulated.This indicated that the inhibition of LY294002to PI3K/Akt signalingpathway was enhanced. However, p-Akt and p-PDK1protein level of three AML cellsco-cultured with HS-5was up-regulated,while p-PTEN was down-regulated.Thisshowed that this pathway of AML cells was activated.And this activation wassuppressed by LY294002.The protein level of p-AktSer47was down-regulated treatedwith LY294002combined with As2O3in AML cells co-cultured with HS-5.This resultindicated that LY294002inhibited the proliferation of AML cells co-cultured withHS-5combined with As2O3by PI3K/Akt signaling pathway.Conclusion:LY294002that is PI3K/Akt signaling pathway inhibitor increased thesensitivity to As2O3of acute myeloid leukemia cells co-cultured with bone marrowstromal cell HS-5.