Investigation On TRAIL As A Reversal Agent For Drug Resistance Of Jurkat Cells Mediated By BMSCs Adhension

Background: Multi-drug resistance continues to be one of the major obstacles hindering the successful treatment of many forms of cancer is to be solved urgently. Recently, researchers have found that cell mediated drug resistance caused by adension of tumor cells to microenvironment plays a critical role in the drug resistance of hematopoietic malignancies,which offers a survival advantage to the tumor cells following initial drug exposure and makes the tumor cells resid in the tumor microenvironment and may facilitate the acquisition of acquired drug resistance.Thus,the tumor microenvironment of hematopoietic malignancies is a novel breakthrough to reverse MDR. The reported CAM-DR models were constructed with normal BMSCs and BMSCs line. Because the BMSCs from leukemia patient are different in adhesion molecule,extracellular matrix and cytokines from normal BMSCs. Furthermore, BMSCs is a cell population consisted of different cells.Thus,we propose that CAM-DR model constructed with BMSCs separated from the leukemia sufferer can imitate the microenvironment of the leukemia cell more really. Based on the mechanisms of CAM-DR found in that model constructed with BMSCs from leukemia sufferer,we selected tumor necrosis factor- related apoptosis-inducing ligand as the tool to reverse the drug resistance and investigated the usefulness and possibilities.Consequently,these results will to be as theories and experiments references for the later investigations on CAM-DR.Objectives:1. To construct the CAM-DR model with the cultured BMSCs from leukemia patients and evaluate the influences of the main microenvironment elements involed in the model to the chemotherapeutics sensitivity. More specifically, we will investigat the changes of the apoptosis modulins and cell cycle to explain the possible mechanisms involved in CAM-DR. 2.To identify the validity of TRAIL to reverse the CAM- DR and try to elucidate the possible mechanism. 3.To evaluate the application prospect of TRAIL to overcome CAM-DR through investigating its influences on normal hematogenesis.Methods: 1.To construct the CAM-DR model with the cultured BMSCs fromleukemia patients,we co-cultured Jurkat cells with the irradiated BMSCs layer by adhesion. In order to confirm the morphous and function features of the model,we abserved the relationship between the Jurkat cells and BMSCs in the model with ligt microscope and scanning electron microscope and analyzed the apoptosis, intracellular drug accumulation and IC50 with FACScan machine and MTT, respectively. 2.To elucidate the possible mechanisms involved in CAM-DR,we analyzed cell cycle progression and the expression of the proteins associated with cell cycle and apoptosis with FACScan machine and Western blot, respectively.Then we used the acquired mechanisms as standard to select TRAIL as a regent to reverse CAM-DR. 3.To determine the expression changes of DR4( TRAIL- Rl) and DR5( TRAIL- R2) in the Jurkat following DNR exposure,we analyzed them at different time points by RT-PCR, the immunofluorescence and FACScan machine. Consequently, the expression of DR5( TRAIL- R2) in the Jurkat can be up-regulated by DNR exposure for 6h. To acquire synergistic effect between DNR and TRAIL,we pretreated Jurkat cells in the CAM-DR model with DNR before TRAIL exposure.To assess the reversal effect of TRAIL on CAM-DR,we analyzed apoptosis and fold reversal(FR) by FACScan machine and MTT dye,respectively.4. To elucidate the reversal mechanisms on CAM-DR of TRAIL, we analyzed the main effect molecules in the apoptosis pathway of TRAIL and modulins associated with apoptosis or cell cycle by FACScan machine and Western blot, respectively. 5.We analyzed the expression and location of DcRl(TRAIL-R3) and DcR2(TRAIL-R4) on normal bone marrow mononuclearcells and BMSCs by immunofluorescence and FACScan machine,respectively.Based on the expression of decoy receptor,we evaluated the influence of TRAIL on the amplification of bone marrow mononuclearcells supported by BMSCs.To determine the toxic effect of TRAIL on normal hematogenesis,we analyzed the number of CFU-GM from mononuclearcells and fluorescence colony from Jurkat marked by GFP with gene transfer technique after following TRAIL exposure.Results: 1. We observed the CAM-DR model constructed with the cultured BMSCs from leukemia patients with scanning electron microscope and found that most of Jurkat were anchored to the "niche" of BMSCs layers and a few Jurkat cells were encapsuled by BMSCs. AS is similar to the relation between leukemic cells and BMSCs in the bone marrow particle from leukemia patients. 2.We identified the function of the model and found that the Jurkat cells in the model showed a decreased sensitivity to DNR,IC50 valuesfor normal BMSCsJeukemic BMSCs and noadhered contol were of 1.78 u mol/L, 2.30 u mol/L and 0.45 u mol/L, respectively.Moreover,Jurkat cells adhered to BMSCs have a survival advantage over suspended cells following DNR exposure for 24h, apoptosis percentages for normal BMSCs,leukemic BMSCs and noadhered contol were of 8.48% ± 0.86%, 6.05% ±0.54% and 25.74% ±6.15%.As compared with with contol, normal BMSCs and leukemic BMSCs had significant difference in IC50 values and apoptosis percentages ( p<0.01).On the other hand, the IC50 value and apoptosis percentage for leukemic BMSCs group were of significantly different from the normal BMSCs group.Interestingly, intracellular drug accumulation in adhered Jurkat cells was not significantly different from the suspended cells( p>0.05).Similar observations that fluorescene signals from DNR were distributed diffusely in both adhered Jurkat cells and suspended cells were made when the cells exposed to DNR were observed under fluorescent microscope. 3.We identified the roles of the main microenvironment elements in the constructed CAM-DR model.As compared with the suspended cells,the Jurkat cells adered to FN and the fixed BMSCs displayed drug resisitance phenotype .Conditioned medium produced from normal BMSCs,leukemic BMSCs and coculture system of Jurkat-normal BMSCs could not protect the Jurkat cells from DNR-induced cell death.But the conditioned medium from the coculture system of Jurkat-leukemic BMSCs could partly protect the Jurkat cells from DNR-induce cell death,IC50 value and apoptosis percentage for this conditioned medium were significantly different from the control(p<0.05). 4. The leukemia BMSCs adhesion up-regulated the levels of Bcl-2, XIAP and c-FLIPLin the Jurkat cells.In the meantime,the level of survivin was down-regulated, but the levels of Bax and c-FLIPs were not changed.The mentioned results indicated that CAM-DR was associated with disequilibrium of antiapoptotic molecules and proapoptotic molecules. In addition, the cell cycle of Jurkat cells in the CAM-DR model was blocked at Gl phase by up-regulation of cyclin D2 and p27,down-regulation of cyclin A, cyclin E and cdk4. Compared with control/The Jurkat cells in the CAM-DR model had 48.74%±8.77% in G0/G1, compared with 27.83%±1.86% in G1/G0 of the suspended Jurkat cells. 5. We detected both mRNA and protein of DR4 and DR5 in the Jurkat . Exposure to 0.5umol/L DNR for 6h could up-regulate DR5 in the Jukat cells from both control group and CAM-DR ,but don't affect the DR4.Therefore, the Jurkat cells in the CAM-DR model hadthe architecture base for TRAIL to reverse drug resistance.6. Treatment with 0.5 u mol/L of DNR for 6 hours followed by 200ng/ml of TRAIL for 18 hours could decrease the IC50 value of CAM-DR model from 2.30 u- mol/L to 0.049 u mol/L.Consistently, treatment with DNR followed by TRAIL could increase apoptosisi percentage from 6.08% ±0.62% to 82.00 % ± 4.25 % .Consequently,TRAIL could reverse CAM-DR efficiently by its synergistic effect with DNR.7. The BMSCs adhesion could partly repress the depolarization of mitochondrium membrane potential and release of cytochrome c from mitochondrium induced by DNR and TRAIL. But the synergistic effect between TRAIL and DNR could weaken the depression effect on mitochondrium pathway for apoptosis.As a consequence, mitochondrium pathway to induce apoptosis could be activated.On the other hand,TRAIL could activate caspase-3 directly through a pathway independent of mitochondrium pathway to induce apoptosis.8.We detected the expressions of DcRl and DcR2 in the BMSCs and bone marrow mononuclearcells,but did not observe the up-regulation of DcRl and DcR2 in the BMSCs and bone marrow mononuclearcells following DNR exposure. Consequently,we did not observe either a evident apoptosis-induced effect or synergistic effect between TRAIL and DNR on BMSCs and bone marrow mononuclearcells to result in apoptosis. Exprements involving mixture of bone marrow mononuclearcells and Jurkat cells marked by GFP showed a marked decrease in the number of fluorescence colonies from the marked Jurkat cells after 24h incubation with 200ng/ml of TRAIL.However,no significant decrease in the number of CFU-GM colonies was detected.These results suggest that TRAIL offers the possibility of being used as an agent to reverse CAM-DR in hematologic malignancies.Conclusions:l.The CAM-DR model contructed by the cultured BMSCs from leukemia suffer can simulate the microenvironment of leukemic cells in vivo.2. As main elements of leukemic microenvironment in the CAM-DR modeljurkat cells-BMSCs interaction, ECM and the cytokines play roles in CAM-DR, and affect mutually, inseparable. 3. The leukemia BMSCs adhesion up-regulated the levels of Bcl-2, XIAP and c-FLIPL in the Jurkat cells.In the meantime,the level of surviving was down-regulated,but the levels of Bax and c-FLIPs were not changed.These results indicated that CAM-DR was associated with disequilibrium of antiapoptotic molecules and proapoptotic molecules. In addition, the accumulation in Gl phase of Jurkat cells in the CAM-DR model also plays a role in celladhesion-mediated drug resistance. 4. Exposure to DNR can up-regulate DR5 and increase the percentage of Jurkat cells in premitotic phases(Gl,S or G2).In the meantime,the levels of survivin and XIAP were down-regulated.As a result, synergistic effect between TRAIL and DNR was displayed. 5. The BMSCs adhesion could partly repress mitochondrium pathway.associated with apoptosis induced by TRAIL.But the synergistic effect between TRAIL and DNR could weaken the depression effect on mitochondrium pathway.As a consequence, mitochondrium pathway to induce apoptosis could be activated.On the other hand,TRAIL could activate caspase-3 directly through a pathway independent of mitochondrium pathway to induce apoptosis. 6. TRAIL can eradicate Jurkat cells efficiently without significant toxity to normal BMSCs and bone marrow mononuclearcells.In other words,TRAIL does not impact normal haemopoiesis.So we propse that TRAIL offers the possibility of being used as an agent to reverse CAM-DR in hematologic alignancies.