| Research background: Acute myeloid leukemia(AML)is a hematological malignant tumor.About 60%-80% of patients have undergone anthracyclines represented by daunorubicin(DNR)combined with cytarabine(Ara-C).The DA(3+7 regimen)regimen can achieve complete remission after induction chemotherapy.However,patients who have not received allogeneic hematopoietic stem cell transplantation(HSCT),only 20%of them can achieve long-term disease-free survival,and many AMLs relapse eventually and have drug resistance.They become refractory AMLs.Patients with refractory and relapsed AML are often resistant to commonly used anthracycline chemotherapy regimens.Therefore,finding new targeted drugs combined with traditional anthracycline chemotherapeutics as a new treatment plan is the current research focus.Studies have shown that the activation of the PI3K/AKT/m TOR signaling pathway affects the survival of tumor patients,and the PI3K/AKT/m TOR signaling pathway is continuously activated in 50%-70% of AML patients.So inhibitors of this pathway may become the key to the treatment of tumors.The previous study of our group found that the m TOR inhibitor rapamycin can reverse the resistance of the adriamycin-resistant strain HL60/ADM to adriamycin and increase the rate of apoptosis.The rapamycin analogue everolimus(RAD001,Afinitor),as a new oral m TOR inhibitor,blocks signal transduction by inhibiting m TORC1 and is active against many types of solid tumors.Compared with rapamycin,the pharmacokinetics of everolimus is more superior.Everolimus is currently used for advanced renal cell carcinoma,subependymal giant cell astrocytoma,and neuroendocrine tumors.The role of everolimus in hematological tumors is rarely reported.Therefore,this article studies the role and molecular mechanism of everolimus and daunorubicin in leukemia.Objective: To study the effect and molecular mechanism of m TOR inhibitor everolimus combined with daunorubicin on myeloid leukemia cells.Methods:1.CCK8 method was used to detect the cell proliferation after everolimus alone or in combination with the chemotherapy drug daunorubicin treated HL60 and HL60/ADM cell lines.HL60 cells was treated with different concentrations of daunorubicin(12.5,25,50,100,200 n M)and everolimus(2.5,5.0,10,20,40,60 μM)for 24 h,48h,72 h.HL60/ADM cells were treated with different concentrations of daunorubicin(78.125,156.25,312.5,625,1250,2500 n M)and everolimus(6.25,12.5,25,50,100μM)for 24 h,48h,72 h.We Calculated the median inhibitory concentration(IC50)and the combination index(CI),finding the appropriate drug concentration for western blot of the combination of the two,and observed whether everolimus combined with daunorubicin could increase the sensitivity of HL60/ADM cells to the drug.2.Annexin-V flow cytometry was used to detect cell apoptosis.We chose everolimus with different concentration gradients of 10,20,40 μM in HL60 cell line and 5,10,20,40,80 μM in HL60/ ADM cell line.We detected the effect of drug treatment on cell apoptosis before and after treatment.3.Western-blot was used to detect the effect of everolimus alone or in combination with daunorubicin on the expression of PI3K/AKT/m TOR signaling pathway molecules in HL60 and HL60/ADM cells.We detected the expression changes of m TOR-related ribosomal protein S6 kinase,apoptosis and anti-apoptosis-related proteins such as BAX,BCL-2,PARP,and Caspase family proteins.Results:1.Inhibitory effects of everolimus and daunorubicin on HL60 and HL60/ADM cells were time-dose dependent.Everolimus down-regulates the IC50 of daunorubicin on HL60 and HL60/ADM cells,and the two had a synergistic effect.(1)CCK-8 was used to detect the IC50 of HL60 and HL60/ADM cells for 48 h of daunorubicin,which was 66.96±19.05 n M and 711.67±74.74 n M.Compared with HL60,the resistance index of HL60/ADM exceeded 10.HL60/ADM is a daunorubicin-resistant strain.(2)The IC50 of HL60 and HL60/ADM cells tested by CCK-8 of everolimus for 48 h was 16.23±1.49 μM and 33.00±4.09 μM,respectively.The resistance index of HL60/ADM to everolimus was 2,indicating that HL60/ ADM also has certain resistance to everolimus.(3)After CCK-8 detection of everolimus(10 μM)combined with daunorubicin on HL60 cells for 48 h,the IC50 of daunorubicin dropped to 7.99±2.40 n M,which was significantly down-regulated(P<0.05).(4)After everolimus(20 μM)combined with daunorubicin treated HL60/ADM cells for 48 h,the IC50 of daunorubicin dropped to 137±3.61 n M,which was significantly down-regulated(P<0.0001).(5)We Calculated the combined drug index of HL60 and HL60/ADM respectively to be less than 1,indicating that everolimus and daunorubicin have a synergistic effect on killing cells.Everolimus significantly down-regulated the IC50 of daunorubicin in HL60 and HL60/ADM cells(P<0.05),and the combination index(CI)was less than1,and the two had a synergistic effect.After everolimus(10μM)combined with daunorubicin treated on HL60 cells for48 h,the IC50 of daunorubicin dropped to 7.99±2.40 n M(P<0.05).After everolimus(20 μM)combined with daunorubicin treated on HL60/ADM cells for 48 h,the IC50 of daunorubicin dropped to 137±3.61 n M(P <0.0001).We calculated that the combination index of HL60 and HL60/ADM was less than 1,which indicated that everolimus and daunorubicin have a synergistic effect on killing cells.2.Everolimus can increase the apoptosis of AML cells in a dose-dependent manner.The results of Annexin V detection of cell apoptosis showed that the apoptosis rate of HL60 and HL60/ADM cells increased with the increasing of the drug dose of everolimus after 48 h.3.Everolimus combined with daunorubicin can promote HL60 and HL60/ADM cell apoptosis by regulating PI3K/AKT/m TOR signal,and increase the sensitivity of AML cells to daunorubicin drugs.The results of Western blot showed that after HL60 and HL60/ADM cells were treated with daunorubicin alone for 48 hours,the PI3K/AKT/m TOR signaling pathway showed that the expression of BAX protein was up-regulated in the two cell lines,and there was no significant difference in the expression of other proteins.Used alone After everolimus acted on HL60 and HL60/ADM cells for 48 hours,the PI3K/AKT/m TOR signaling pathway showed m TOR,P-m TOR,RPS6 downregulation,and BAX up-regulation.Everolimus(10 μM)combined with daunorubicin(0.025 μM)treated HL60 cells for 48 h,PI3K,AKT,m TOR,P-m TOR,RPS6,and Bcl-2 were all down-regulated,and the down-regulation effect was greater than that of everolimus or daunorubicin alone.BAX and Cleaved-PARP were all up-regulated,Casepase3 changed little.In HL60/ADM cells,after everolimus(20 μM)combined with daunorubicin(0.4 μM),PI3 K,AKT,P-AKT,m TOR,P-m TOR,RPS6,Bcl-2were all down-regulated,and the down-regulation effect was greater than that of everolimus or daunorubicin alone.BAX and Cleaved-PARP were all up-regulated,and Casepase3 had little change.Conclusion:1.Everolimus combined with daunorubicin synergistically kills HL60 and HL60/ADM cells,activates the PI3K/AKT/m TOR signaling pathway,and increases the apoptosis of HL60 and HL60/ADM cells.2.Everolimus combined with anthracyclines is a way to clinically reverse the drug resistance of relapsed and refractory leukemia. |
PDF Full Text Request