The Study On Influence Of Imatinib-Induced Apoptosis In Chronic Myeloid Leukemia Cells Co-culture With Fibronectin Adhesion

Background and ObiectiveFor the past four decades even though the treatments of leukemia have made great progress,the drug-resistant,residual,refractory and relapse remains the important obstacles to the cure of leukemia today.Therefore,it is necessary to further study how the leukemia cells come out into the drug-resistant phenotype.Traditionally,drug resistance mechanisms have been identified and functionally characterized in unicellular models.Implicit in these unicellular models is the lack of consideration of host-tumor cell interactions that may participate in the emergence of the drug-resistant phenotype.Increasingly,it appears that,like hematopoietic stem cells,leukemic stem cells---those cells responsible for the maintenance and resurgence of leukemia after elimination of the bulk of the leukemic blasts-interact with nonhematopoietic cells of the bone marrow microenvironment.Collectly,the bone marrow microenvironment is considered as a tumor sanctuary and contributor to drug resistance,hence the phrase "environment-mediated drug resistance"(EM-DR). Increasing evidences have pointed to the critical role of cell-cell interaction and that the relationship between leukemic cells and the microenvironment correlates with the minimal residual disease.But how and what they crosstalk with each other is still open to debate. As a general studies showed,integrin-mediated adhesion to extracellular matrix (ECM) components fibronectin,collagen,vitronectin,osteopontin,laminin,or stromal cells via cell receptors such as vascular cell adhesion molecule-1 induces signaling critical for the regulation of survival/apoptosis,proliferation,cell-cycle progression,cell shape,polarity,adhesion,migration and differentiation of normal and tumor cells.Interactions of cells with their microenvironment modify the cellular sensitivity for radiation- and drug-induced cellular injury.Recently,integrin-mediated adhesion was shown to improve leukemia cell survival after exposuring to cytotoxic drugs orγ-irradiation damage and contribute to drug resistance.Evidence is mounting thatβ1 integrin may be a molecular therapeutic target for tumour.In previous studies,we explored wether the expression ofβ1 integrin and focal adhesion kinase is correlated with the disease deterioration and poor prognosis.We found that,the expression ofβ1 integrin on bone marrow mononuclear cells in CML patients with blast crisis was significantly higher than that in CML patients with chronic-phase,conversely,patients with CML-blast crisis had lower expression of focal adhesion kinase.At the same time,we found that the mRNA level of Rho(a small GTPase) gene was up-regulated in imatinib-resistant K562 cells by microarray analyses and the Rho protein up-regulated in CML patients with blast crisis by proteomic analyses.Futher,we detected the expression of RhoA in CML patients with imatinib-resistant and explore the functions of RhoA in imatinib-resistant K562 cells.We have shown that the expression of RhoA mRNA and protein was found in both imatinib-sensitive and imatinib-resistant patients,but the later show higher expression.Similarly,the over-expression of RhoA was found in imatinib-resistant K562 cells.When the RhoA gene was knocked down,imatinib-resistant K562 cells had shown significant G0/G1 arrest,the expression ofβ1 integrin and the percentage of apoptotic increased.Considered that ABL kinase domain mutations of P210BCR-ABL is the most prevalent mechanism of resistance to Imatinib.Hence, nested reverse transcriptase-polymerase chain reaction was performed on imatinib-sensitive and imatinib-resistant K562 cells to amplify the ABL kinase domain,followed by direct sequencing and sequence homologous analysis.But we had not found ABL kinase domain mutations of P210BCR-ABL in imatinib-resistant K562 cells.So,we speculate that there is some unknown signals leading K562 cells resistantanc to imatinib other than the ABL kinase domain mutations.Focal adhesion kinase and Rho are the important kinases in the integrin signal, and base on our data,we hypothesis that integrin/FAK/Rho pathway coduct the critical signaling between leukemic cells and the microenvironment,finally leading to leukemia cells resistantanc to imatinib.In order to address this issue,we construct the matrix and leukemia cells co-culture model and investigate the influences of bone marrow microenvironment the major components-fibronectin in the co-culture system on Imatinib sensitivity of leukemia cells.Then applying the functional anti-β1 integrin antagonistic monoclonal antibody to the co-culture system,further explore the activity of downstream protein FAK and Rho proteins with or without integrin activation.We propose that The importance of integrin/FAK/Rho pathway for drug resistance makes these molecules promising drug targets for the prevention of drug resistance induced by the bone marrow microenvironment.Methods1.Treating the K562 cells with Imatinib at different doses and time,we perform a MTT assay experiment to compare the cell proliferation inhibition rate of each group,to determine the best deal conditions. 2.K562 cells would be inoculated with fibronectin-coated plate(Fn),collagen-coated plate(Co) and suspended cultures as control (mask),then a MTT assay experiment would be performed to compare the OD value of each group,to calculate the adhesion rate both of Fn and Co group,to determine the appropriate cellular density of each group.3.K562 cells would be inoculated with Fn,Co and mask group,then treated with Imatinib at different doses and time,a MTT assay experiment performed to compare the cell proliferation inhibition rate of each group,to explore the protection from Imatinib of K562 cells adhesion to Fn.4.K562 cells would be inoculated with Fn,Co and mask group,then treated with Imatinib at different doses,48 hours later a AnnexinV-FITC/PI assay experiment performed to compare the cell apoptosis rate of each group,to explore the protection from Imatinib of K562 cells adhesion to Fn.5.Bone marrow mononuclear cells from CML patient would be inoculated with Fn, Co and mask group,then treated with Imatinib at different doses and time,a MTT assay experiment performed to compare the cell proliferation inhibition rate of each group,to explore the protection from Imatinib of bone marrow mononuclear cells adhesion to Fn.6.K562 cells would be inoculated with Fn,then Pull down-Wersten Blot performed to detecte Rho-GTP content changes over time.Further K562 cells would be inoculated with Fn,Co and mask group,treated with anti-integrin and without as control,48 hours later Pull down and Wersten Blot performed to detecte Rho-GTP, p-FAK content.7.Statistical Methods:The datas from exploring the best deal conditions and cell proliferation inhibition assay were analyzed by repeated measures ANOVA. The datas from comparing the Apoptosis assay were analyzed by two-way ANOVA.The datas from measuring the adhesion rate and Western Blot experiments were analyzed by one-way ANOVA.SPSS13.0 software used for data processing,statistical analysis.Results1.K562 cells treating with imatinib at dose levels 0.0μM,0.4μM,0.8μM,1.6μM,3. 2μM,6.4μM and time 0h,24h,48h,72h,the datas from MTT assay show,the cell proliferation inhibition rate among the different doses of imatinib(F=117.592,P= 0.000) and the different time(F=194.981,P=0.000) both have significant difference.2.K562 cells were inoculated with Fn,Co and mask group,the MTT assay performed 24 hours later,and the average OD value of 3 were 0.53±0.14,0.61±0.17,1.03±0.20 (F=21.643,P=0.000),further found that Fn Vs.mask,Co Vs mask both had significant differences,but Fn Vs Co had no significant difference.According to the absorbance ratio(adhesion/mask ),the adhesion rates of Fn and Co were 51.16%and 59.11%,respectly.3.K562 cells were inoculated into Fn,Co and mask group with the time 24 hours,48 hours and 72 hours,the data from MTT assay experiment had shown that,all the main effect of groups had significant difference(P value of 0.006,0.000 and 0.000 respectly),further found that Fn Vs mask and Fn Vs mask both had significant differences,but mask Vs Co had no significant difference;the main effect of Imatinib concentrations all had significant difference(P value of 0.000,0.000 and 0.000 on the time 24h,48h and 72h respectly).4.K562 cells were inoculated with Fn,Co and mask groups,treated with Imatinib at doses 0.8μM and 1.6μM,the AnnexinV-FITC/PI assay experiment performed to compare the cell apoptosis rate of each group 48 hours later,the data had shown, the total apoptotic cells at 0.8μM dose level in each group rate(%) were 16.24±3.47,26.05±3.44,30.55±6.43,respectly(F=7.378,P=0.024);at 1.6μM dose level were 35.33±1.63,48.91±3.72,55.26±6.31,respectly(F=16.528,P=0.004). Further analyzed found that Fn Vs Co and Fn Vs mask both had significant differences,the apoptosis rate of Fn was significantly lower than Co,mask groups. The apoptosis rate of Co Vs mask had no significant difference.5.Bone marrow mononuclear cells from patients with chronic myeloid leukeimia were inoculated into Fn,Co and mask group with the time 24 hours,48 hours and 72 hours,the data from MTT assay experiment had shown that,all the main effect of groups had significant difference(P value of 0.006,0.000 and 0.000 respectly), further found that Fn Vs mask and Fn Vs mask both had significant differences,but mask Vs Co had no significant difference;the main effect of Imatinib concentrations all had significant difference(P value of 0.000,0.000 and 0.000 on the time 24 hours,48 hours and 72 hours respectly).6.K562 cells were inoculated with Fn,then Pull down-Wersten Blot performed to detecte Rho-GTP content on the time 24h,48h and 72h.Results showed that,the ratio of Rho-GTP/GAPDH were 0.4546±0.0179,0.8729±0.0614,0.8654±0.0905,0.8362±0.0101,respectly(F=13.230,P= 0.002).the Comparison of the Rho-GTP content among on the time 24 hours,48 hours and 72 hours had significant differences(P=0.000);But the Comparison of the Rho-GTP content each two among the time on 24 hours,48 hours and 72 hours all had no significant differences.7.K562 cells were inoculated with Fn,Co,mask,Fn+anti CD29 mAb,Co +anti CD29 mAb and mask+anti CD29 mAb,group,24 hours later Pull down and Wersten Blot performed to detecte Rho-GTP,p-FAK content.Results showed that, the ratio of p-FAK/GAPDH were 1.4067±0.2463,0.7971±0.1283,0.9724±0.2713, 1.0236±0.1274,0.2266±0.0241,0.5610±0.0437,respectly(F=17.588,P=0.000);the ratio of Rho-GTP/GAPDH were 1.0513±0.3920,0.3500±0.0624,0.5174±0.1099, 0.7579±0.2109,0.3277±0.1006,0.4246±0.0625,respectly(F=6.280,P=0.004). Further analysis show that,the Comparison of the Rho-GTP content for Fn Vs mask and Fn Vs Co both had significant differences;Intresting,we found that, there is significant differences in the Comparison of the p-FAK content for the Fn group treated with anti-integrin comparing to without;but there is no significant differences in the Comparison of the Rho-GTP content for the Fn group treated with anti-integrin comparing to without.Conclusions1.compared to the collagen adhesion co-culture,either K562 cells lines or CML patients with bone marrow mononuclear cells,fibronectin adhesion co-culture can be significantly reduced imatinib-induced cell growth inhibition and apoptosis,we cosider that interaction between leukemic cells and the bone microenvironment components fibronectin contribute to Imatinib resistance.2.Fibronectin adhesion co-culture reduced imatinib-induced cell growth inhibition and apoptosis,and this may be correlated to Rho-GTP activity.The anti-integrin monoclonal antibody block the p-FAK levels decreased to the control group,but not the Rho-GTP levels.This suggesting that anti-integrin monoclonal antibody can not completely block the K562 integrin cell surface molecules and fibronectin or the effective ntegration of K562 cells and another pathway activated Rho protein.