ERK Is Involved In The MSC-induced Drug Resistance Of T-cell Acutelymphoblastic Leukemia Cells

Acute leukemia have been obtained significant progress in the treatment and targeted therapies at the present stage,it still a poor prognosis in patients with leukemia.In recent years,with the deepening of the bone marrow microenvironment research,find that the bone marrow(BM)stromal niche can protect acute leukemia cells against the cytotoxicity of chemotherapeutic agents and is a possible main reason of relapse.So the study of blocking the signal pathway of bone marrow niche between leukemia cells become the target of resistant to reverse and improve the prognosis.Similar to normal hematopoietic cells,leukemia cells need to adhere to the bone marrow niche for proliferation,survival,self-renewal,differentiation and chemoresistance.Better understanding of the molecules which regulate leukemia cells retention to the BM and their dissemination to the circulation will help us to prevent the relapse of the leukemiaIn recent years,researchers have started to intervene interaction pathways between leukemia cells and bone marrow niche so as to achieve increased sensitivity to Chemotherapeutic drugs,and found that ERK signaling pathway plays an important role in this process.Blocking ERK signaling pathway can reduce the adhesion of the bone marrow niche quiescent leukemia cells and enhance cell cycle dependent cytotoxic chemotherapy drugs,so as to eliminate minimal residual disease(MRD),reduce relapse.SCH772984 is a specific inhibitor of the ERK signaling pathway,whether it can block the leukemia cells and bone marrow stromal adhesion in vitro study,whether it can affect proliferation and apoptosis of leukemia is the purpose of this research.We create bone marrow mesenchymal stem cells and leukemia cell lines co-culture system in vitro,After SCH772984 block the ERK signaling pathway,we detect cell cycle and apoptosis of leukemia cells and changes in leukemic cells to chemotherapeutic drug sensitivity.Clarify the biological characteristics of leukemic cells by block the ERK signaling pathway,provide a theoretical basis for theelimination of the bone marrow microenvironment new treatment MRD and reverse multidrug resistance.Objectives:1、 Discussion the the ERK signaling pathway Whether it will affect the cell cycle and Ara-c induced apoptosis of Jurkat cells which co-cultured with MSCs(Jurkat + MSCs)or without(Jurkat)MSCs and treated with or without SCH772984.2、 Western blot analysis ERK1/2、p-ERK1/2 expression of Jurkat cells which co-cultured with(Jurkat + MSCs)or without(Jurkat)MSCs.Methods:1.CCK-8 assay was used to determine the IC50 of Jurkat cell lines.2.Apoptosis and cell cycle were detected by FCM.3.Western blot analysis ERK1/2、p-ERK1/2 expression in Jurkat cells.Results:1、Cytarabine for Jurkat cell lines,concentration of the cell lines of work were 0.01 mg/ml.2、FCM detecte the Ara-c induced apoptosis of Jurkat cells which co-cultured with MSCs(Jurkat + MSCs)or without(Jurkat)MSCs and treated with or without SCH772984,1)without(Jurkat)MSCs culture group:There was no significant differences of apoptosis of Jurkat cells between the treated with or without SCH772984 group.Also,be similar to between Ara-c treated group and Ara-c associate with SCH772984 treated group(P>0.05);2)co-cultured with(Jurkat + MSCs)MSCs group:Compared with without(Jurkat)MSCs culture group,apoptosis rate was significantly decreased in the adhesion culture group(P <0.05).3、FCM detecte the cell cycle of Jurkat cells which co-cultured with MSCs(Jurkat + MSCs)or without(Jurkat)MSCs and treated with or without SCH772984,1)without(Jurkat)MSCs culture group:There was no significant differences of G0/G1 、 S phase of Jurkat cells between the treated with or without SCH772984 group.(P>0.05);2)co-cultured with(Jurkat + MSCs)MSCs group:Compared with without(Jurkat)MSCs culture group,Jurkat cells in the adherent culture group G0 / G1 phase no significant change,G2 / M phase fraction less,increase the percentage of S phase(P <0.05);3)co-cultured with(Jurkat + MSCs)MSCs group:Jurkat cells treated with or without SCH772984 Compare,treated with SCH772984 group G0 / G1 phase no significant change,S phase reduced,increase the percentage of G2/M phase.4、Western blot analysis ERK1/2、p-ERK1/2 expression of Jurkat cells which co-cultured with(Jurkat + MSCs)or without(Jurkat)MSCs,highest ERK1/2、p-ERK1/2 expression of Jurkat cells which co-cultured with(Jurkat + MSCs).Conclusions:1、BM-MSCs reduce the Ara-c induced apoptosis of Jurkat cells by adhering culture.The ERK signaling pathway inhibitor SCH772984 enhanced Jurkat cells sensitivity to chemotherapy.2、Bone marrow mesenchymal stem cell mediated cell cycle arrest of Jurkat cells,main arrest in Sphase.The ERK signaling pathway inhibitor SCH772984 can promote the growth of Jurkat cells by blocking Jurkat cells adhered to BM-MSCs.3、Western blot analysis ERK1/2、p-ERK1/2 expression of Jurkat cells which co-cultured with(Jurkat + MSCs)or without(Jurkat)MSCs,highest ERK1/2、p-ERK1/2 expression of Jurkat cells which co-cultured with(Jurkat + MSCs).