Vincristine Reversion Of The Bone Marrow Mesenchymal Stem Cells On Leukemia Asparaginase Resistance

BackgroundWith the research of new mechanisms and targets of anticancer drugs, the treatmentof childhood leukemia is progressing. However, the emergence of drugs resistance andeven multidrug resistance, impedes the chemotherapy of childhood leukemia. In thegrowth of leukemia, the interaction between leukemic cells with the bone marrowmicroenvironment has become an important part to study the role of bone marrowmicroenvironment. Through the secretion of cytokines and adhesion to leukemic cells,mesenchymal stem cells play an important role in the proliferation, differentiation,migration, and apoptosis of leukemic cells. The treatment of leukemia need not onlyeliminate the leukemia cells, but also destroy the abnormal microenvironment which theleukemia cells survive. With the combination of L-asparaginase (L-asp) treament,complete remission rate and long-term disease-free survival of children with acutelymphoblastic leukemia (ALL) have been significantly improved. As a first-line drug inchildhood ALL chemotherapy, ALL L-asp has an irreplaceable position in remissioninducion in childhood ALL. However, it is easy to develop drug resistance, there is adirect correlation between the resistance to L-asp and poor clinical efficacy. As theresistance mechanisms of L-asp, have not yet unified, but some studies have shown thatthey may relate to the high expression of ASNS in mesenchymal stem cells (MSCs),which are in the bone marrow microenvironment. Embark on interference and cutting offthe interaction between ALL cells and MSCs, it is likely to find more solutions to theproblem of asparagine resistance, and improve the prognosis of ALL. All childrennowadays, the sequential therapy of vincristine (VCR) and L-asp are used in childhoolALL chemotherapy. The principle of the sequential therapy is that VCR may contribute tothe inhibition of L-asp allergic reactions, and studies have found that MSCs are sensitiveto antimicrotubule drugs, for instance VCR. Whether the pretreatment of MSCs with VCR,can reduce the expression of ASNS in MSCs and reverse the L-asp resistanse whichmediated by MSCs in co-culture system or not, it remains to be confirmed.On the basis of these studies, the study simulate the model of leukemia cells in bonemarrow microenvironment in vitro, and build the different co-culture systems, that is theco-culture systems with or without L-asp treatment, and with or without VCRpretreatment the MSCs. By comparisons the apoptosis of leukemia cells and expression levels of ASNS mRNA in leukemia cells between different culture systems, to assess theimpact of MSCs on L-asp sensitivity. Explore whether VCR can inhibit the protectiveeffect of MSCs on leukemia cells during L-asp treatment or not and the possiblemechanism, to lay the foundation for further understanding the mechanisms of ALL cellsresistance to L-asp and overcome it.Objective1. Isolate, culture and amplify childhool acute lymphoblastic leukemia bone marrowprimary cells in vitro，then identify them.2. Explore the effects of MSCs to leukemia cell line Jurkat on asparaginase resistanceand the mechanisms.3. Pretreat MSCs with VCR, and explore their effects to leukemia cell line Jurkat onasparaginase resistance and the mechanisms.Methods1. Isolate and amplify childhood ALL bone marrow primary cells in vitro.2. Test leukemia cell lines (Jurkat、HL-60) IC50(50%inhibition rate) by MTT assay，aftertreated in different concentrations of asparaginase,and establish co-culturedMSCs-Jurkat system. Detect the apoptosis rate of Jurkat in different co-culture groupsand separate cultured groups by flow cytometry.3. Detect ASNS mRNA expression levels of Jurkat cell line in different treatedco-culture groups and separate cultured groups by RT-PCR.Technology roadmap Results1. Successfully isolate，culture and amplify childhood ALL MSCs in vitro, thenestablish the different drugs treatments co-culture system.2. Compared to HL-60, Jurkat are more sensitivity to L-asp. Identify the IC50ofJurkat to L-asp.3. The effect of co-culture and different drugs treatments on Jurkat apoptosis rateand ASNS mRNA expression. 3.1Impact of L-asp on Jurkat cell line: Jurkat cell line with L-asp treated, itsapoptosis rate and ASNS mRNA expression are increased(P <0.05); co-culturegroup with L-asp treated (co-culture group2), compared to co-culture group1, theapoptosis rate and ASNS mRNA expression of Jurkat cell line both increase（P<0.05）.3.2Effects of MSCs on Jurkat cell line: The anti-apoptotic capacity of Jurkat cellline in co-culture group1is stronger than separate culture group (P <0.01), ASNSmRNA expression elevate (P <0.05); The anti-apoptotic capacity of Jurkat cellline in co-culture group2is stronger than separate culture group with L-asptreated (P <0.01), but ASNS mRNA expression has no significant change.3.3Effects of MSCs with VCR pretreated on Jurkat cell line: Pretreat MSCs withVCR,then establish co-cultured group3,Jurkat cell line apoptosis rate inco-culture group3are higher than co-culture group2(P <0.01),，while ASNSmRNA expression has no obvious change.Conclutions1. With L-asp treated separate culture group and co-culture group, both canincrease Jurkat cell line apoptosis rate and ASNS mRNA expression, but thechange of apoptosis and ASNS mRNA expression is lower in co-cultured groupthan separate culture group. The result improve that even in the support ofMSCs, L-asp still have effects on Jurkat cell line, MSCs can partiallysuppressed L-asp cytotoxicity.2. Jurkat cell line co-cultured with MSCs can enhance its anti-apoptotic ability andreduce ASNS mRNA expression.3. MSCs pretreated with VCR, can enhance the anti-apoptotic ability of Jurkat cellline in co-culture group, but its ASNS mRNA expression has no significantchange.