| Background/ObjectivesChronic myeloid leukemia(CML)is a severe hematologic malignancy characterized by uncontrolled proliferation of pluripotent hematopoietic stem cells.About 95%CML patients express the BCR-ABL fusion protein,which has persistent tyrosine kinase activity.Abnormal signals of BCR-ABL tyrosine kinase activate downstream targets,reprogram cells and allow them to multiply uncontrollably,which leads to myeloid hyperplasia.Tyrosine kinase inhibitors(TKIs)is the most effective drug for molecular targeted therapy of chronic myelogenous leukemia.With the widespread use of TKIs in clinical practice,patients also exhibit tolerance to second-and even third-generation TKIs.The transcription factor subfamily(FOXO1,FOXO3,FOXO4,FOXO6)of the forkhead box class O(FOXO)serves as a downstream target for the PI3K-AKT signaling cascade pathway.The results show that FOXO3a mediates the effects of antitumor chemicals through PI3K-AKT,ERK,JNK and p38 signaling pathways,and can regulate the expression of mdr-1/P-gp,an enhanced drug resistance genes.It is also of great significance in regulating the sensitivity and tolerance of antitumor drugs.Human umbilical cord derived mesenchymal stem cells(UC-MSCs)can be derived from umbilical cords treated as medical wastes.Thus,invasive surgery is not used to gain them,in which condition the ethical issue in the use of embryonic stem cells is avoided.Moreover,these cells have low immunogenicity and good amplification capacity,which makes them be widely studied and used in recent years.The application of UC-MSCs in the treatment of tumors and other diseases has been extensively studied.However,there are few studies on the effects of UC-MSCs on the tolerance of tumor cells to chemotherapeutic drugs and molecularly targeted drugs.Based on the above,this project intends to establish imatinib-resistant K562 cell line for human leukemia and study its drug resistance and possible molecular mechanisms.The MSCs were isolated and prepared from fresh umbilical cord tissue and identified by surface molecular markers and multilineage differentiation capabilities.By co-cultivating UC-MSCs with imatinib-resistant K562 cells and parental K562 cells respectively,the interactions between UC-MSCs and leukemia cells and their drug susceptibility were studied,and the possibility of using UC-MSCs to reverse the resistance of leukemia was discussed which can provide new ideas for the clinical intervention of leukemia-targeted drug resistance.Methods1.Establishment of imatinib K562/R cell line for human leukemiaStarting from low concentration,the drug resistance of human leukemia K562 cells(designated as K562/R cells)is inducted by the method of increasing the imatinib induced concentration in long-term run with different phases.MTS assay was used to detect the sensitivity of K562 parental cells and K562/R cells to imatinib,as well as the cross-resistance to other anti-tumor drugs.The expressions of P-gp and BCR-ABL were detected by Western-blot.The flow cytometry was used to detect the level of apoptosis of K562 cells and K562/R cells which were induced by imatinb.2.Primary isolation,culture and identification of UC-MSCsUC-MSCs were isolated by tissue block adherence from aseptic fresh human umbilical cord tissue.The morphology of the cells obtained by the separation was observed under an inverted microscope to determine whether they conformed to the cell morphology of the MSCs.Flow cytometry was used to detect the surface molecular markers of CD34,CD44,CD45 and CD90isolated from MSCs.The prepared MSCs were evaluated by adipogenic,osteogenic and neuron-like directed differentiation.3.Detection of drug resistance of leukemia cells co-culture with UC-MSCsThe isolated and identified UC-MSCs were co-cultured with established K562/R cells and K562 parental cells respectively.K562/R cells and K562 cells cultured separately were used as the respective control cells.MTS assay was used to detect changes of sensitivity of K562/R cells and K562 cells to imatinib respectively after co-culture with UC-MSCs for 48,72 and 96 hours.RT-PCR was used to detect the drug resistance of K562/R cells and K562 cells and the expression of such apoptosis-related genes like mdr-1,CD44,FOXO3a,Bax and Caspase-3 before and after co-culture.Western-blot was used to detect the expression of P-gp,CD44,FOXO3a,Bax,Caspase-3 and other proteins before and after co-culture.The apoptosis rates of co-cultured cells were detected by flow cytometry.4.Changes of the content of leukemia stem cells in leukemia cell populations after co-cultured with UC-MSCsUC-MSCs were co-cultured with K562/R cells and K562 cells respectively,and then K562/R cells and K562 cells cultured separately were used as the respective control cells.The flow cytometry was used to detect the changes of the content of leukemia stem cells in K562/R cell and K562 cell populations before and after co-cultured with UC-MSCs.5.Interference of short hairpin RNA silences FOXO3a gene expression in K562/R cellsA short hairpin RNA interference plasmid targeting the FOXO3 gene was constructed,and then lentivirus packaging was performed in HEK293T cells,and then K562/R cells were infected with FOXO3 lentivirus(sh-FOXO3)cells.Finally,stably silenced cells(K562/R-shFOXO3)were obtained by screening culture.The cell proliferation activity and drug sensitivity were detected by MTS assay.The apoptosis rate was detected by flow cytometry and the expressions of FOXO3a,P-gp and CD44 were detected by Western-blot.6.Changes of PI3K/AKT and MAPKs pathways in K562/R before and after co-cultured with UC-MSCsUC-MSCs were co-cultured with K562/R cells and K562 parent cells respectively,and then K562/R cells and K562 cells cultured separately were used as the control group cells.Western-blot was used to detect the changes of proteins expression of AKT,PI3K,p38,phosphorylated p38,phosphorylated ERK,phosphorylated c-jun,c-abl and phosphorylated c-abl in leukemia cells before and after co-cultured with UC-MSCs.Results1.Establishment and characteristics of imatinib-resistant K562 cell lineStarting from low concentration,the drug resistance of human leukemia K562 cells is inducted by the method of increasing the imatinib induced concentration in long-term run with different phases.Then the half inhibitory concentrations(IC50)of K562 and K562/R cells against imatinib are measured by MTS assay.The results showed that after 24 h,48 h and 72 h of imatinib treatment,the multiplication resistance of K562/R cells was 64.22,40.52,31.58 times of K562cells respectively,and expressed cross-tolerance with such conventional chemotherapy drugs as ADM,PXL,MMC,THP,CHOEP,HHT and AMN107.After treated with imatinib to K562 and K562/R cells for 24 h,Annexin V-FITC and PI double staining showed that the apoptosis rate of K562/R was lower than that of K562 cells,suggesting that K562/R cells have a strong anti-apoptotic effect on imatinib.After 48 h treatment with imatinib,K562 cells had such morphological changes as cell shrinkage and disintegration,whereas no obvious morphological changes were observed in K562/R cells.Western-blot results showed that BCR-ABL and P-gp protein increased its expression level as the increase of concentration and time.The above results prove that with long-term induction of imatinib,a stable drug-resistant K562/R cell line with multidrug resistance and resistance to imatinib has been successfully established.2.Isolation,culture and identification of UC-MSCsUC-MSCs were prepared from sterile umbilical cords obtained after full-term caesarean delivery of volunteers.The UC-MSCs were successfully isolated and obtained by tissue block method for 2-3 weeks.Under the inverted microscope,the cells showed whirlpool-like fibrous cell morphology.Flow cytometry detected the expression of CD44 and CD90 on the surface of UC-MSCs,but the hematopoietic stem cell marker CD34 and human common leukocyte antigen CD45 were not expressed,which accorded with the phenotypic criteria of UC-MSCs.Targeted differentiation showed that the isolated cells were able to differentiate into adipocytes,bone cells and neuron-like cells,which meets the criteria for in vitro induced multilineage differentiation of UC-MSCs.Through the identification of morphological analysis,cell surface immunophenotypic phenotype and in vitro induction of multi-directional differentiation,it was confirmed that UC-MSCs cells were successfully isolated and prepared from umbilical cord tissue.3.UC-MSCs reverse the tolerance of leukemia cells to imatinib and doxorubicinUC-MSCs were directly co-cultured with imatinib-resistant cell line K562/R and K562parental cell line,respectively,and K562/R and K562 cells cultured separately were used as respective control cells.Under the inverted microscope,both K562 and K562/R cells began to show adhesion to UC-MSCs,fusiform deformation and fusion into UC-MSCs after 48 h.MTS assay showed that the sensitivity of K562/R to imatinib and doxorubicin increased after co-culture.The expressions of such apoptosis-related genes as Bax and Caspase-3 increased at the 48 h,72 h and 96 h after co-culture.The expression of such resistance-related genes as mdr-1,CD44 and FOXO3a was decreased respectively after the co-culture.Flow cytometry showed that the apoptosis rate of co-cultured K562/R cells induced by imatinib was higher than that of K562/R cells cultured alone.Western-blot results showed that the expression of apoptosis-related proteins as Caspase-3 and Bax increased after K562/R cells co-cultured with UC-MSCs;co-cultured K562/R cells show decreased levels of resistance-associated proteins as P-gp,CD44 and FOXO3a.The above results fully confirmed that UC-MSCs can reverse the resistance of K562/R cells to imatinib and doxorubicin.4.UC-MSCs reverse resistance of K562/R cells by reducing leukemia stem cells in K562/R cell populationUC-MSCs were directly co-cultured with imatinib-resistant cell line K562/R and K562parental cell line for 48 h,72 h and 96 h,respectively,and K562/R and K562 cells cultured separately were used as respective control cells.CD34~+CD38~-were used to be markers of leukemia stem cells,and using flow cytometry,we analyzed the change of leukemia stem cells in K562/R and K562 cell population.Compared with control cells,the content of Leukemia stem cells derived from K562/R had a significant reduction.However,in K562 cell population,the content of Leukemia stem cells had a slight decrease.These results showed that UC-MSCs can reduce the content of Leukemia stem cells to sensitize K562/R cells.5.UC-MSCs down-regulate FOXO3a/Pgp axis to sensitize K562/R cells by modulating PI3K/AKT and MAPK/JNK pathwayTo study the mechanism of UC-MSCs reversal of K562/R cell resistance and whether FOXO3a is involved in the regulation of UC-MSCs reversal of drug resistance,shRNA interference was used to silence the expression of FOXO3 in K562/R cells.It found that K562/R cells are significantly more sensitive to imatinib.After 48 h of treatment of imatinib,the IC50decreased and the apoptosis rate increased.Western-blot results showed that after FOXO3 was effectively silenced,the expression levels of P-gp and CD44 of K562/R cells were also significantly reduced.UC-MSCs were directly co-cultured with imatinib-resistant cell line K562/R and K562 parental cell line for 48 h,72 h and 96 h,respectively,and K562/R and K562 cells cultured separately were used as respective control cells.Western-blot analyzed the expressions of key proteins in PI3K/AKT and MAPK/JNK parthway.The results showed that the expressions of PI3K and AKT remarkably decreased in a time-dependent manner in K562/R and K562 cells.Also,phospholyted c-Jun had a reduction only in K562/R cells.However,the protein level of p38 had no difference at different time point,and that of phospholyted p38 and phospholyted-ERK increased after co-cultured with UC-MSCs.Combined with above results that the expression of such resistance-related proteins as P-gp,CD44 and FOXO3a was decreased in K562/R cells after co-cultured with UC-MSCs,our results suggest that UC-MSCs maybe regulate PI3K/AKT and MAPK/JNK parthway to reduce the expression of FOXO3a,and finally lead to decrease in the expression of P-gp,CD44,which reverse resistance of K562/R cells.Conclusions1.A stable drug-resistant K562/R cell line with multidrug resistance and resistance to imatinib has been successfully established with the long-term induction form low to high concentration gradually.2.UC-MSCs cells with MSC morphological features and surface markers were successfully isolated and prepared from fresh human umbilical cord tissue using tissue block adherence.Moreover,the cells have multi-directional differentiation ability and can be in vitro stably amplified.3.UC-MSCs can effectively enhance the sensitivity of K562/R cells to imatinib and doxorubicin and reverse their resistance with direct contact with K562/R cells.The mechanism may be related to the decrease of FOXO3a expression in K562/R after co-cultured with UC-MSCs by modulating PI3K/AKT pathway and MAPK/JNK pathway,which further inhibits the expression of resistance-related proteins like P-gp and CD44. |
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