The Effect And Mechanism Of Histone Deacetylase Inhibitors On The PKC412 Resistant AML Cells

BackgroundAcute myeloid leukemia(AML)is the most common acute leukemia among adult patients,new cases have been rising by average 2.2%annually over the past decade.During 2003 to 2012,mortality had been rising by 0.2%per year and in 2012 alone about 4.4 per 100,000 people diagnosed acute myeloid leukemia.According to statistical reporting,from 2000 to 2011,5-year survival rate was only 25.9%.Due to the chemotherapy treatment induced drug resistance,the relapse rate of AML is high.Therefore,in order to gain more effective treatment of AML,it's very necessary to looking for more effective treatment strategies for AML to against the drug resistance.FLT3(FMS-like tyrosine kinase 3)belongs to the class ? receptor tyrosine kinase family,occurring in approximately 89%of AML patients.Recent studies showed that mutations in FLT3 occur in about 30%of AML patients and few ALL patients.The most frequently FLT3 mutation type,in about 20-25%of AML patients and 5%of MDS patients,represents an internal tandem duplication(ITD)in the juxtamembrane region.What's more,there are approximately 7%patients has point mutation with the "activation loop",which most occurred in D835Y,less commonly is N841I and Y842C.So far,some studies showed that the point mutations in the juxtamembrane of FLT3 also can activated FLT3.The mutation of FLT3 can constitutively increases FLT3 autophosphrylation and activated the signaling pathways of PI3K-AKT?RAS-MEK-MAPK and STAT5,resulting in uncontrolled cell proliferation and blockage of cell differentiation.Base on the recent researches,the existing mutation of FLT3-ITD has a significant relation to poor prognosis.These results indicated FLT3 is an appropriate therapeutic target.In recent years,scientific research workers have developed a variety of FLT3 inhibitors,such as CEP-701?PKC412?MLN518?SU11248?ABT-869 and Sorafenib,which has entered different stages of clinical trails.Results showed that FLT3 inhibitors can reduce the ratio of peripheral blood and bone marrow cells and delay disease progression.However,in the course of application of these inhibitors,some patients had rapid emergence of drug resistance,which seriously limits the clinical application of FLT3 inhibitors.Thus,recognizing the drug resistance of FLT3 inhibitors will help us to find more suitable treatment strategies,to prevent or reverse the drug resistance.It's the key to successful treatment of AML with FLT3 targeted therapy.Studies had showed that there are many mechanisms underlying drug resistance of FLT3 inhibitors,including acquired point mutation,overexpression of FLT3,drug resistance gene,sustaining activated proliferative signaling,expression of anti-apoptotic molecules and protection of bone marrow micro-environment.For the complication of drug resistance of FLT3 inhibitors,researchers developed new inhibitors,applied combination therapy and FLT3 antibody and other methods to overcome drug resistance.But,results showed that all these methods can not completely reversing drug resistance and gain better clinical responses.Moreover,in these studies,researchers showed that when gaining the drug resistance to FLT3 inhibitors,patients often got the cross drug resistance.Histone deacetylase inhibitor(HDACi)has been proved to be a kind of effective anti-cancer drugs of hematological malignancies.Studies had confirmed that HDACi and cytosine arabinoside combination can enhance the sensitivity of AML cells to cytarabine,they has synergetic effect.Therefore,whether HDACi plays the same role on the FLT3 inhibitor resistant cells?Until now,there is no researche about the relationship of HDACi and FLT3 inbibitor resistance.In order to discuss the effect of HDACi on the PKC412 resistance,we constructed a PKC412 resistant MV4-11 cell and tested the biological characteristics of drug resistance cell,explored the possible underlying drug resistance mechanism.Then we applied HDACi to the drug resistant cells and explored the effect and possible molecular mechanism,providing new strategies for clinical treatment.Methods1.The effect of PKC412 on the biological activity of AML cellsMV4-11(FLT3-ITD)and THP-1(wt-FLT3)cells were used in our experiments.MV4-11 cells were treated with different concentration(0,0.01,0.02,0.04,0.08,0.16,0.32uM)of PKC412,THP-1 cells were treated with different concentration(0,0.01,0.02,0.04,0.08,0.16,0.32,1,2,4,8,16,32uM)of PKC412 for 24,48,72h,the proliferation of cells was estimated by MTS.2?The effect of HDACi on the biological activity of AML cellsMV4-11(FLT3-ITD)and THP-1(wt-FLT3)cells were used in our experiments.MV4-11 cells and THP-1 cells were respectively treated with different concentration(0,1,2,4,8,16,32,64nM)of LBH589,different concentration(0,0.1,0.25 0.4,0.8,1.6,3.2uM)of SAHA for 24,48,72h,the proliferation of cells was estimated by MTS.3?The effect of HDACi and PKC412 on cell proliferation of MV4-11 cellsMV4-11(FLT3-ITD)cells were used in our experiments.MV4-11 cells were respectively treated with different concentration(3,6,12nM)of LBH589,different concentration(0.2,0.4,0.8uM)of SAHA,different concentration(5,10,20nM)of PKC412,different concentration(3nM+5nM,6nM+10nM,12nM+20nM)of LBH589+PKC412,different concentration(0.2uM +5nM,0.4uM+10nM,0.8uM+20nM)of SAHA+PKC412 for 72h,the proliferation of cells was estimated by MTS.4?The single or combination effect of PKC412,LBH589 and SAHA on cell cycle and apoptosis of MV4-11MV4-11(FLT3-ITD)cells were used in our experiments.MV4-11 cells were respectively treated with PKC412(40nM),LBH589(16nM),SAHA(1.6uM),PKC412+LBH589(40nM+16nM),PKC412 +SAHA(40nM+1.6uM)24h,cell cycle and apoptosis were estimated by flow cytometry.5?Inducing MV4-11 cells resistant to PKC4125.1 The experimental procedure of inducing MV4-11 cells resistant to PKC412We used three methods to induce MV4-11 cells resistant to PKC4121)MV4-11 cells were cultured with a starting dose of PKC412(6.6 nM)cell culture media,then constantly increased until the dose of PKC412(130nM).2)MV4-11 cells were cultured with a starting dose of PKC412(1 nM)cell culture media,then constantly increased until the dose of PKC412(130nM).3)MV4-11 cells were cultured with a starting dose of PKC412(1 nM)cell culture media,then cell viability reach 90%,we double the dose of PKC412.5.2 The effect of PKC412 on cell proliferation of MV4-11-R cellsMV4-11-R cells were used in our experiments.MV4-11-R cells were treated with different concentration(0,0.01,0.02,0.04,0.08,0.16,0.32uM)of PKC412 for 24,48,72h,the proliferation of cells was estimated by MTS.6?The effect of PKC412 on cell cycle,apoptosis and FLT3 signaling pathways of MV4-11 and MV4-11-R cellsMV4-11 and MV4-11-R cells were used in our experiments.MV4-11 and MV4-11-R cells were treated with different concentration(0,40nM)of PKC412,cell apoptosis and cell cycle were estimated by flow cytometry.The phosphorylation and total level of FLT3 protein was estimated by western blot between MV4-11 and MV4-11-R cells.MV4-11 cells were treated with different concentration(0,20,80nM)of PKC412,MV4-11-R cells were treated with different concentration(0,80,320nM)of PKC412,the protein level of p-FLT3,total FLT3 and other related proteins were detected by Western blot.7?The effect of HDACi on cell proliferation,the protein level of FLT3,cyclin of MV4-11-R cellsMV4-11-R cells were used in our experiments.MV4-11-R cells were treated with different concentration(0,1,2,4,8,16,32,64nM)of LBH589,different concentration(0,0.1,0.2,0.4,0.8,1.6,3.2uM)of SAHA for 24,48,72h,the proliferation of cells was estimated by MTS.MV4-11-R cells were treated with different concentration(0,8,32nM)of LBH589,different concentration(0,0.8,3.2uM)of SAHA,the protein level of FLT3 and other related proteins were detected by Western blot.Statistical MethodsThe data of results were represented as the mean,standard deviation and using IBM SPSS 20 sofeware package.T-test was performed to compare the different between two groups.Differences among groups were determined using One-way ANOVE.Survival rates were determined using analysis of variance of factorial design.P values less than 0.05 are considered statistically significant.Results1.The comparison of different effect of PKC412 on MV4-11 and THP-1 cellsPKC412 can inhibit the proliferation of MV4-11 cells,and this effect was dependent on density and time.We found the 24h,48h,72h IC50 of PKC412 on MV4-11 cells respectively were 61.27±5.73,40.09±2.38,22.52±0.6(nM).The concentration of PKC412 was more than luM can significantly inhibit the proliferation of THP-1 cells,and this effect was dependent on density and time.The 24h,48h,72h IC50 of PKC412 on MV4-11 cells respectively were 6.76±0.46,3.12±0.17,1.48±0.2(uM).The effect of PKC412 on MV4-11 was significantly stronger than that of THP-1 cells,there was a significant statistical difference.(p<0.05)2.The comparison of different effect of LBH589 and SAHA on MV4-11 and THP-1 cellsLBH589 can inhibit the proliferation of MV4-11 cells,and this effect was dependent on density and time.We found the 24h,48h,72h IC50 of LBH589 on MV4-11 cells respectively were 29.51±1.04,17.02±1.44,10.51±1.95(nM).We finded out when the concentration of LBH589 was lower than 8nM,LBH589 had no inhibit effect on THP-1 cells,when it came to 16nM,LBH589 can obviously inhibit the proliferation of THP-1 cells.The 24h,48h,72h IC50 of LBH589 on MV4-11 cells respectively were 53.47±1.23,38.43±0.81,31.35±0.31(nM).The effect of LBH589 on MV4-11 was significantly stronger than that of THP-1 cells,there is a significant statistical difference.(p<0.05)SAHA can inhibit the proliferation of MV4-11 and THP-1 cells,and this effect was dependent on density and time.We found the 24h,48h,72h IC50 of SAHA on MV4-11 cells respectively were 1.59±0.09,1.12±0.06,0.68±0.05(uM).The 24h,48h,72h IC50 of SAHA on THP-1 cells respectively were 3.09±0.43,2.61±0.36,1.57±0.09(uM).Comparing with the two drugs,SAHA was more effectively on MV4-11 cells than THP-1,especially high concentration,there was a significant statistical difference.(p<0.05)3.The combined effect of LBH589,SAHA and PKC412 on MV4-11 cellsLBH589 and PKC412 has synergistic effect on MV4-11 cells,the CI value was less than 1,what's more,with the increasing concentration,more obvious synergy.SAHA and PKC412 also has synergistic effect and as then same as LBH589 and PKC412.4.The single and combined effect of LBH589,SAHA and PKC412 on the cell apoptosis and cell cycle of MV4-11 cellsPKC412 and LBH589 both can induce cell apoptosis in MV4-11 cells,especially in early apoptosis.Compared with the control group,the early and late apoptosis phase of cells in PKC412 group were increased,there was a significant statistical difference.(p<0.05).Compared with the LBH589 and control group,we can find the early apoptosis phases cells of LBH589 was significantly increased(p<0.05),however,the late apoptosis phase cells had no difference.When the two drugs combined,PKC412+LBH589 group compared with control group,the early and late apoptosis phase cells in PKC412+LBH589 group increased more dramatically.(p<0.05)5.Inducing the drug resistant MV4-11-R to PKC412 and the comparison effectof PKC412 on MV4-11 and MV4-11-R cells.5.1 Inducing the drug resistant MV4-11-R to PKC412We cultured the MV4-11 cells in the culture medium with the increasing concentration of PKC412,after 4 months,we conducted PKC412 resistant MV4-11 cells.The 24h,48h,72h IC50 of PKC412 on MV4-11-R cells respectively are 389.66±15.51,313.31±73.59,198.73±16.80(nM),was about 9 times more than MV4-11 cells.5.2 The comparison effect of PKC412 on MV4-11 and MV4-11-R cellsAt the concentration of 10nM,20nM,40nM,80nM,160nM,320nM,the cell survival rate of the drug resistant MV4-11-R to PKC412 was significantly higher than that of PKC412 sensitive cells,there was a significant statistical difference.(p<0.05)5.3 The comparison of PKC412 on the cell apoptosis and cell cycle of MV4-11 and MV4-11-R cellsPKC412 can induce cell apoptosis in MV4-11 cells,especially in early apoptosis,had a statistical difference(p<0.05).However,to MV4-11-R,the same concentration of PKC412 had no effect on the early and late apoptosis phase cells,there was no significant statistical differenceIt was the same with the cell cycle,PKC412 can induce MV4-11 cells arrest on cell G1 phase(p<0.05).However,the same concentration of PKC412 had no effect on the early and late apoptosis phase of MV4-11-R cells,what's more,there was no statistical difference between the G1,S phase cells compared with control group.5.4 The effect of PKC412 on the FLT3 and downstream signaling pathway molecular of MV4-11 and MV4-11-R cellsWe found that the level of P-FLT3 and T-FLT3 of the MV4-11 and MV4-11-R had no difference.PKC412 can inhibit the phosphorylation of FLT3 of MV4-11 cells,also it can inhibit the phosphorylation level of downstream ERK,AKT and STAT5.And for MV4-11-R cells,we found that high concentration of PKC412 also can inhibit FLT3 phosphorylation,but the phosphorylation levels of downstream of ERK,AKT and STAT5 had no significantly decrease.6.The effect of LBH589 and SAHA on the FLT3 and downstream signaling pathway molecular of MV4-11-R cellsLBH589 and SAHA can inhibit the proliferation of MV4-11-R cells,and this effect dependent on density and time.We found the 24h,48h,72h IC50 of LBH589 on MV4-11-R cells respectively were 20±0.99,11.55±0.67,7.76±0.64(nM).The 24h,48h,72h IC50 of SAHA on MV4-11-R cells respectively were 0.89±0.05,0.64±0.05,0.55±0.03(uM).We found out that LBH589 and SAHA act on MV4-11-R cells,not only the phosphorylation levels of FLT3,ERK,AKT and STAT5 were significantly decrease,but also the total levels of FLT3,AKT and STAT5 were decreased.We also find that,after LBH589 and SAHA act on MV4-11-R cells,the cyclin P21was also significantly increased.Conclusion1.PKC412 can inhibit FLT3 positive AML cells,and especially the FLT3 mutation cells.2.LBH589 and SAHA can inhibit FLT3 positive AML cells,and especially the FLT3 mutation cells3.LBH589,SAHA and PKC412 can separately or had synergy effect on FLT3 mutation cells,with the higher concentration,the more obvious synergies.4.PKC412,LBH589 and SAHA can induce the cell apoptosis and cell cycle arrest of MV4-11,and the synergy effect is more significant.5.PKC412 drug resistant MV4-11-R cells can activate the FLT3-independent cell proliferation signaling pathway,when the phosphorylation levels decrease,the phosphorylation level of ERK,AKT,STAT5 was not significantly reduced.6.LBH589 and SAHA can inhibit MV4-11-R cells,the mechanism may be related to the decrease of P-ERK,P-AKT,P-STAT5,and elevated level of P21.