Study On The Role Of 4EBP1-eIF4E-Mcl-1 Axis In The Synergistic Killing Of AML Cells In Microenvironment By HHT And ATO

Objective:To study the role of the 4EBP1-eIF4E-Mcl-1 axis in HHT and ATO killing co-cultured AML cells,and to deeply analyze the mechanism of action of HHT and ATO killing adherent co-cultured AML cells.Methods:1.To construct a subcutaneous tumor-bearing model of human AML mice,and to explore the mechanism of homoharringtonine and arsenic trioxide killing leukemia cells in vivo;2.Co-culture AML cells with human bone marrow stromal cells HS-5 to establish an in vitro co-culture system in order to simulate the bone marrow microenvironment that leukemia cells depend on;3.Detect the drug sensitivity of AML cells by CCK-8 method and Annexin V-APC/7AAD double staining method;4.Using Western Blot method to detect the expression of 4EBP1-eIF4E-Mcl-1 axis-related protein before and after co-cultivation;5.After expression and knockdown of eIF4E,the changes in sensitivity to HHT and ATO were detected;6.Using molecular biological inhibition methods against 4EBP1,eIF4E and Mcl-1,we further explore the role of 4EBP1-eIF4E-Mcl-1 axis in killing AML cells by HHT and ATO.Results:1.In vivo studies in mice showed that HHT can cooperate with ATO to clear HL-60 leukemia cells in vivo,significantly inhibit the growth of tumor volume in tumor-bearing mice,and significantly reduce tumor load;and detected by immunofluorescence,4EBP1-eIF4E-Mcl-1 axis key protein p-4EBP1^Thr37/Thr46,p-eIF4E^ser209 and Mcl-1 expression decrease under the action of HHT and ATO.2.Co-culture can significantly reduce the chemotherapeutic sensitivity of AML cells to HHT and ATO.After co-culture,the expression of p-eIF4E^Ser209,Mcl-1,p-4EBP1^Thr37/Thr46and p-Mnk1^{Thr197/Thr202} was enhanced,while eIF4E,4EBP1 and Erk1/2 expression were not change significantly.The treatment of HHT and ATO alone can significantly inhibit the expression of p-eIF4E^Ser209,Mcl-1,p-4EBP1^Thr37/Thr46 and p-Mnk1^{Thr197/Thr202}.3.Overexpression of eIF4E inhibits the chemotherapy sensitivity of AML cells,while knocking down eIF4E enhances the chemotherapy sensitivity of AML cells.4.After using the specific inhibitors of 4EBP1-eIF4E-Mcl-1 signal axis related proteins,it was further verified that HHT and ATO exert a killing effect on AML cells through the 4EBP1-eIF4E-Mcl-1 axis.Conclusion:HHT and ATO in vivo can kill AML cells and exert anti-leukemia effect.The 4EBP1-eIF4E-Mcl-1 axis is involved in the killing of AML cells by HHT and ATO,and inhibiting the activation of this axis is conducive to enhancing the chemotherapy sensitivity of AML cells.