The Roles And Mechanisms Of Chemotherapy-induced DNA Damage To BMSCs In AML Chemo-resistance And The Aberrant Expressions Of Th Cells In AML

Section I Screening of BMSCs DNA damage secreted proteins and identification of their roles in AML chemo-resistanceBackground:Acute myeloid leukemia (AML) is a group of hematopoietic malignant disease characterized by genetic abnormalities and great differences of response to treatment and prognosis, seriously threatening the human health. Up to now, although the anthracycline-based and cytarabine-based combined chemotherapy, new targeted agents and hematopoietic stem cell transplantation haved partly improved the survival of patients with AML, most of patients eventually suffer from recurrence and death caused by multi-drug resistance (MDR), indicating a poor prognosis. Therefore, there are important clinical significances to investigate the mechanisms of AML chemo-resistance and search for the countermeasures to improve the curative effect, reduce recurrence and improve outcomes of patients with AML.Recently, an increasing number of studies have shown that the interaction of the bone marrow (BM) microenvironment with leukemia cells plays an important role in the MDR and recurrence of leukemia. The BM microenvironment is mainly composed of bone marrow stromal cells (BMSCs), extracellular matrix and a variety of soluble factors, in which BMSCs are the most important factors in the survival of leukemia cells and drug resistance. Studies have reported that BMSCs reduced the drug sensitivity of HL60 and multi-resistant HL60/VCR cells to topotecan (TPT) through regulation of Bcl-2 and Caspase 3. Another study showed that bone marrow endothelial cells can promote the proliferation and suppress apoptosis of AML cells induced by chemotherapy drugs. Our previous study showed that AML cells had an effect on the proliferation, migration and tube formation of the bone marrow endothelial cells through direct contact, which is involved in the drug resistance of leukemia cells. Thus, BMSCs can improve leukemia drug resistance through various mechanisms and targeting BMSCs maybe the powerful means to overcome the AML drug resistance.Recent studies suggest that chemotherapy drugs not only induce DNA damage in tumor cells leading to cell death or senescence, but also can induce genotoxic stress in stromal cells of the tumor microenvironment, initiating the DNA damage response and activating secretion of the downstream cytokines, which play an important role in mediating acquired drug resistance of tumors. Studies found that chemotherapy induced obvious DNA damage in prostate cancer fibroblasts, secreted a spectrum of increased proteins and promoted prostate cancer cells proliferation and drug resistance through secretion of WNT16B and activation of classical Wnt signaling pathways. Another study showed that adriamycin can induce DNA damage in mice thymus endothelial cells and initiate the stress response, secreting factors that affected the survival of lymphoma cells, such as IL-6 and TIMP-1. Others showed that after chemotherapy damage, endothelial cells and stromal cells can reduce the sensitivity of the breast cancer cells to chemotherapy drugs through secretion of TNFa and activation of the NF-κB signaling and CXCL1/2 expression. Recent studies have shown that chemotherapy drugs can also induce damage to the BMSCs in the BM microenvironment at the same time of killing the leukemic cells. However, Which proteins are secreted by damaged BMSCs and how are the leukemia chemo-resistance mediated by the secreted proteins? ALL above remains unknown and requires further investigation.Objectives:The aim of this study was to determine that chemotherapy can induce DNA damage to BMSCs and umbilical vein endothelial cells (HUVECs) in the BM microenvironment and identify the roles of conditioned medium from damaged BMSCs and HUVECs in AML drug resistance. Additionally, we will further screen and verify differentially expressed proteins in the conditioned medium from damaged BMSCs through human antibody array and perform the biological functions and downstream signaling pathways analysis using associated software. Based on the results of antibody array, we will then identify the pivotal factors among BMSCs DNA damage secretory proteins in regulation of AML chemo-resistance in vitro and investigate their expressions in the BM of AML patients. Our original research will reveal the new mechanisms of the BM microenvironment in AML drug resistance, which has a great significance to over the AML drug resistance and eliminate minimal residual disease.Methods:1. The effect of chemotherapy on BMSCs and HUVECs DNA damage:The bone marrow stromal cell line (HS-5) and HUVECs were respectively exposed to adriamycin (ADR), idarubicin (IDA), cytarabine (Ara-C) and normal saline (NS) for 24 hours. Cell immunofluorescence and Western blot were applied to detect the expressions of y-H2AX and PARP1.2. The effect of the conditioned medium from damaged BMSCs and HUVECs on the biological behaviors of AML cells.2.1 The effect of conditioned medium on the sensitivity of AML cells to chemotherapy drugs:AML cells were cultured with conditioned medium from ADR-treated, IDA-treated and NS-treated HS-5 cells, and RPMI 1640 medium containing 0.5% FBS was chose as controls. Every group was treated with different concentrations of ADR and IDA. The relative survival rate of AML cells was analyzed by CCK8 assay.2.2 The effect of conditioned medium on the proliferation of AML cells:AML cells were cultured with conditioned medium from ADR-treated and NS-treated HS-5 cells, and RPMI 1640 medium containing 0.5% FBS was chose as controls. CCK8 was used to detect the proliferation of AML cells and the growth curve was drawn.3. The effect of direct co-culture HS-5 with AML cells on the apoptosis of AML cells: HS-5 cells were co-cultured with Kasumi-1 cells in different proportion and treated with ADR for 48 hours. Cells were stained with Annexin V/PI and were immediately measured by flow cytometry.4. The expressions of IL-6, IL-8 and CYR61 in damaged BMSCs:ELISA was applied to detect the protein levels of IL-6, IL-8 and CYR61 in conditioned medium from damaged HS-5 cells and primary mesenchymal stem cells (MSCs). Real-time RT-PCR was used to detect the mRNA expressions of IL-6, IL-8 and CYR61 in HS-5 and MSCs after treated with ADR, IDA, Ara-C and NS.5. Screening the differentially expressed proteins in conditioned medium from damaged BMSCs:The human antibody array was used to detect the protein expression profile in conditioned medium from NS-treated and ADR-treated HS-5 cells and the proteins with higher difference multiple were chose as candidate factors.6. Verification of the antibody array:Real-time RT-PCR was applied to detect the mRNA expressions of candidate factors in HS-5 cells after treated with ADR, IDA and Ara-C.7. The analysis and validation of biological functions and signaling pathways of protein array results:We performed the Go Term and signaling pathway analysis using related software. THP-1 and U937 cells were cultured with conditioned medium from IDA-treated and NS-treated HS-5 cells and RPMI 1640 medium containing 0.5% FBS for 6 hours. Western blot was used to identify the analyzed results of signaling pathways.8. The expressions of candidate DNA damage secreted proteins in the BM of AML patients:BM samples were obtained from 144 cases of patients with AML and 34 cases of healthy controls. The bone marrow mononuclear cells (BMNCs) were isolated by density-gradient centrifugation. Real-time RT-PCR was applied to detect the mRNA expressions of candidate proteins.9. Identification of the pivotal factors mediating the AML multidrug resistance among the BMSCs DNA damage proteins:AML cells were hungrily grown overnight and were then cultured in the culture medium with different concentrations of candidate proteins or neutralizing antibodies. Flow cytometry and CCK8 were applied to detect the effect of candidate proteins on apoptosis, proliferation or the drug sensitivity of AML cells.Results:1. Confirmation of Chemotherapy-induced DNA damage to BMSCs and HUVECs: Cell immunofluorescence and Western blot confirmed that y-H2AX and PARP1 were significantly increased in HS-5 and HUVECs after treated with ADR, IDA or Ara-C compared with controls.2. The effect of the conditioned medium from damaged BMSCs and HUVECs on the biological behaviors of AML cells.2.1 The effect of conditioned medium on the sensitivity of AML cells to chemotherapy drug:CCK8 determined that the relative survival rates of AML cells in conditioned medium from damaged HS-5 and HUVECs were significantly increased relative to conditioned medium from HS-5, HUVECs and RPMI 1640 medium after exposure to different concentrations of ADR and IDA.2.2 The effect of conditioned medium on the proliferation of AML cells:CCK8 results showed that conditioned medium from ADR-treated HS-5 cells had very little promoting effect on the proliferation of AML cells.3. The effect of direct co-culture HS-5 with AML cells on the apoptosis of AML cells: The results of flow cytometry showed that co-culture HS-5 with Kasumi-1 cells significantly decreased the apoptosis of Kasumi-1 cells induced by ADR.4. The protein and mRNA expressions of IL-6, IL-8 and CYR61 in damaged BMSCs:4.1 ELISA showed that there was no significant increase of IL-6, IL-8 and CYR61 levels in the conditioned medium from ADR-treated HS-5 and MSCs compared with controls.4.2 Real-time RT-PCR determined that the mRNA expressions of IL-6 and IL-8 were significantly increased in HS-5 cells after treated with ADR and (or) IDA compared with control and no significant change of CYR61 was found. In addition, no significant change of IL-6, IL-8 and CYR61 mRNA expressions was found in MSCs after treated with ADR, IDA and Ara-C compared with control.5. Human antibody array results:We determined that the levels of 44 proteins were significantly increased in the conditioned medium from ADR-treated HS-5 cells. Notably, among these factors, the expressions of Activin A, Betacellulin (BTC), FGF10, EG-VEGF,6Ckine and growth hormone (GH) had the greater increase, which were considered as the candidate factors.6. Verification of the antibody array results:Real-time RT-PCR determined that the mRNA levels of Activin A, BTC, FGF10, EG-VEGF,6Ckine and GH were significantly increased in damaged HS-5 cells compared with controls.7. The analysis and validation of biological functions and downstream signaling pathways of protein array results:GO Term analysis revealed their roles in cell proliferation, differentiation, apoptosis, invasion and signaling transduction. Pathway analysis showed that the DNA damage secreted proteins play their biological functions through cytokine-cytokine receptors interaction, ALK1, TGF-β, PI3K/AKT and JAK/STAT pathways. Western blot results showed that conditioned medium from IDA-damaged HS-5 cells can significantly increase the phosphorylation levels of P38 MAPK, AKT, ERK and STAT3, which is consistent with the results of software analysis.8. The expressions of candidate DNA damage secreted proteins in the BM of AML patients:Real-time RT-PCR determined that the mRNA expressions of Activin, 6Ckine, FGF10, EG-VEGF and GH were significantly increased in AML patients compared with control, and significantly increased in chemo-treated AML patients compared with newly diagnosed AML patients.9. Identification of the pivotal factors mediating the AML multidrug resistance among the BMSCs DNA damage proteins:9.1 The effect of recombinant human Activin A or Activin A neutralizing antibody on proliferation and drug-sensitivity of AML cells:CCK8 results showed that there was no significant change of proliferation and relative survival rate of Kasumi-1 after treated with ADR and IDA in the group containing Activin A compared with controls. CCK8 also determined that the relative survival rate of THP-1 cells had no significant change after Activin A neutralizing antibody treatment when cultured with conditioned medium from IDA-treated and NS-treated HS-5 cells and RPMI1640 medium containing 0.5% FBS.9.2 The effect of recombinant human EG-VEGF on apoptosis, proliferation and drug-sensitivity of AML cells:Flow cytometry and CCK8 results showed that the apoptosis rate and proliferation of THP-1 cells had no significant change after different concentrations of EG-VEGF. Besides, after treated with ADR and IDA, no significant change of relative survival rate of THP-1 cells was found with the increased concentrations of EG-VEGF.9.3 The effect of recombinant human BTC on the apoptosis, proliferation and drug-sensitivity of AML cells:Flow cytometry and CCK8 results showed that the apoptosis rate of THP-1 cells was slightly decreased and the proliferation rate of THP-1 cells was increased with the increased concentrations of BTC. After treated with IDA, the relative survival rate of THP-1 cells was slightly increased with the increased concentrations of BTC compared with controls.9.4 Recombinant human FGF10 enhanced the resistance of AML cells to IDA:CCK8 assays showed that the relative survival rate of THP-1 cells was obviously increased in the group containing after treated with IDA compared with controls.Conclusion:1. Chemotherapy drugs can induce DNA damage to BMSCs and HUVECs, which mediated AML mutidrug resistance through secretion of a spectrum of DNA damage proteins.2. The DNA damage secreted proteins by BMSCs may promote AML multidrug resistance through MAPK and STAT3 signaling pathway.3. The exogenous FGF10 can reduce the sensitivity of AML cells to chemotherapy drugs, indicating that FGF10 may be the pivotal protein in chemo-induced DNA damage to BMSCs mediated AML multidrug resistance.Section Ⅱ The mechanisms of FGF10/FGFR2 signaling in the AML chemo-resistance mediated by chemotherapy-induced DNA damage to BMSCsBackground:Recent studies suggested that the tumor microenvironment endowed tumors with the congenital tolerance to chemotherapy drugs and targeting the tumor microenvironment has become a hotspot in the field of tumors. Chemotherapy, radiotherapy and other stresses can induce DNA damage to benign supporting cells in the tumor microenvironment and initiate the DNA damage response, resulting in the production and secretion of a spectrum of cytokines, growth factors and protease, which promote the inflammation, tumor angiogenesis, epithelial-to-mesenchymal transition and proliferation of tumor cells, and enhance the resistance of tumor cells to chemotherapy drugs, eventually mediating tumor drug resistance. Therefore, it may provide novel target therapy for tumors to explore the roles and mechanisms of DNA damage secreted proteins released by stromal cells of the tumor microenvironment in the tumor drug resistance.Fibroblasts growth factor (FGF) is an important factor in the bone marrow (BM) hematopoietic microenvironment and plays an important role in the hematopoietic regulation, and is closely correlated with leukemia cell proliferation, apoptosis and drug resistance. FGF 10, known as keratinocyte growth factor (KGF2), is one member of FGF families and is mainly secreted by fibroblasts and other mesenchymal cells. Fibroblast growth factor receptors (FGFRs) are the major receptors of FGF, belong to the tyrosine kinase receptor family and are mainly composed of FGFR1, FGFR2, FGFR3 and FGFR4, among which FGFR2 is the high affinity receptor of FGF10. Recent studies have shown that FGF 10 can promote tumor cells proliferation in autocrine and paracrine manners and play an important role in pancreatic cancer, breast cancer, lung cancer, skin cancer, prostate cancer and other solid tumors. Other studies found that FGF10 can promote the proliferation and inhibit the apoptosis of tumor cells through activation of STAT1/P21, MAPK, PLC-y and PI3K pathways. Another study suggested that FGF10 induced pancreatic cancer cell migration and invasion by activation of FGFR2, leading to poor prognosis. The above studies indicate that FGF10/FGFR2 signaling plays an important role in tumor drug resistance and FGF10/FGFR2 signaling is expected to be a new therapy target for overcoming tumor drug resistance.In the study of section one, we identified 44 BMSCs DNA damage secreted proteins through the human antibody array and the expressions of Activin A,6Ckine, FGF10, EG-VEGF and GH with higher change folds were further verified. Meanwhile, we found that the exogenous recombinant human FGF10 can reduce the sensitivity of AML cells to chemotherapy drugs through the biological behavior experiments, indicating FGF10 may be the pivotal factor in chemo-induced DNA damage to BMSCs mediated AML drug resistance. However, the specific regulatory mechanisms remain unclear.Objectives:Our study intends to further explore the mechanisms of FGF10 secreted by damaged BMSCs mediating AML drug resistance based on the study of section one. To investigate the changes of signaling pathways in damaged BMSCs and search for the upstream regulatory mechanisms of FGF10 expressions through intervention of signaling pathways. To determine the expressions of FGFRs in AML cells, the BM of AML patients and the effect of the conditioned medium from damaged BMSCs on the FGFRs expressions in AML cells, and explore the influences of FGF10 on the FGFRs expressions and the downstream signaling pathways of AML cells. To investigate the probability of reversing AML drug resistance through targeting FGF10/FGFR2 signaling using small molecule inhibitors of FGFRs and FGFR2-shRNA lentivirus. Our study will reveal the novel molecular mechanisms of FGF10/FGFR2 signaling in chemotherapy-induced DNA damage to BMSCs mediated the AML drug resistance from the angle of the BM microenvironment and provide new methods for targeted therapy of AML.Methods:1. The change of signaling pathways in damaged BMSCs and HUVECs:HS-5 and HUVECs were respectively exposed to adriamycin (ADR), idarubicin (IDA), cytarabine (Ara-C) and normal saline (NS) for 24 hours. Western blot was used to detect the change of NF-κB, P38 MAPK, mTOR and β-catenin signaling pathways.2. The effect of blocking NF-κB signaling pathway of BMSCs on the biological behaviors of AML cells and the expressions of DNA damage secreted proteins.2.1 NF-κB inhibitors treatment:We used the NF-κB inhibitors Bay 11-7082 to treat HS-5 cells for 1 hour,3 hours and 6 hours. Western blot was used to study the protein expressions of NF-κB P65, p-P65 and IKBa to verify the inhibition effect.2.2 The effect of blocking NF-κB signaling pathways of BMSCs on the drug sensitivity of AML cells and apoptosis:AML cells were cultured in the conditioned medium from HS-5 cells treated with Bay 11-7082 or Bay 11-7082 combined with ADR, and treated with a certain concentration of ADR for 72 hours. CCK8 and Annexin V/PI staining flow cytometry methods were applied to detect the change of relative survival rate and apoptosis rate of AML cells.2.3 The effect of blocking NF-κB signaling pathways of BMSCs on the expressions of DNA damage secretory proteins:HS-5 cells were treated with Bayl 1-7082 for 6 hours followed by treated with ADR, IDA or Ara-C for 72 hours. The mRNA expressions of IL-6, IL-8, CYR61, Activin A,6Ckine, FGF10, EG-VEGF and GH were detected by Real-time RT-PCR.3. Investigation of the mechanisms of FGF10 expression in damaged BMSCs regulated by β-catenin pathway:HS-5 cells were exposed to different concentrations of β-catenin activator (LiCl) for 24 hours. Cell immunofluorescence, Western blot and Real-time RT-PCR were applied to detect the expressions of β-catenin, its downstream target genes (c-Myc, CyclinDl, CD44, Trib2) and FGF10.4. Detection of FGFRs (FGFR1, FGFR2 and FGFR3) expressions.4.1 Real-time RT-PCR was applied to study FGFR1, FGFR2 and FGFR3 mRNA expressions in AML cells.4.2 The expression of FGFRs in the BM of AML patients:BM samples were obtained from 144 cases of patients with AML and 34 cases of healthy controls. Bone marrow mononuclear cells were isolated by density-gradient centrifugation. Real-time RT-PCR was applied to detect mRNA expressions of FGFR1, FGFR2 and FGFR3.4.3 The effect of the conditioned medium from damaged BMSCs on the FGFRs expressions in AML cells:THP-1 cells were cultured with conditioned medium from IDA-treated and NS-treated HS-5 cells and RPMI1640 medium for 24 hours, Real-time RT-PCR was used for detecting FGFR1 and FGFR2 mRNA expressions.5. The mechanisms of FGF10/FGFR2 signaling in AML drug resistance.5.1 The change of FGFR1 and FGFR2 expressions in AML cells treated with recombinant human FGF10:THP-1 and U937 cells were hungrily grown overnight and were treated with different concentrations of FGF 10 for a certain time. Western blot and Real-time RT-PCR were applied to detect the expressions of FGFR1, FGFR2 and downstream activated molecule pFRS2a.5.2 The change of downstream signaling pathways in AML cells after treated with recombinant human FGF 10:After FGF 10 treatment, Western blot was used to detect the phosphorylation levels of P38 MAPK, AKT, ERK and STAT3.6. Investigation the probability of blocking FGFR2 expression to reverse AML drug resistance.6.1 The effect of blocking FGFR2 expression on the drug-sensitivity of AML cells: THP-1 and U937 cells were treated with FGFRs inhibitors BGJ398 and PD173074 for 1 hour followed by treated with ADR, IDA and Ara-C for 72 hours. CCK8 was used to detect the relative survival rate of AML cells.6.2 The effect of blocking FGFR2 expression on the downstream signaling pathways in AML cells:THP-1 and U937 cells were treated with FGFRs inhibitors BGJ398 and PD173074 for 1 hour. Western blot was applied to detect the change of FGFR2, the downstream activated molecule pFRS2a and P38 MAPK, AKT, ERK and STAT3 signaling pathways.7. The effect of down-regulation of FGFR2 expression on the proliferation and drug sensitivity of AML cells.7.1 lentivirus infection:THP-1 cells were infected with FGFR2-ShRNA (ShFGFR2) and Control-ShRNA (ShCtrl) lentivirus and the efficiency of infection was detected withffluorescence microscope after 72 hours. Puromycin was used to gain the stable infection cells. Western blot and Real-time RT-PCR were used to detect the expression of FGFR2 to verify the efficiency of infection.7.2 Cell proliferation assay:CCK8 was used to detect the proliferation of THP-1 cells infected with ShFGFR2 and ShCtrl lentivirus.7.3 Drug sensitivity assay:CCK8 was used to detect the relative survival rate of THP-1 cells infected with ShFGFR2 and ShCtrl lentivirus after treated with different concentrations of ADR.8. The effect of blocking FGFR2 on the expression of tumor suppressor gene PTEN: THP-1 cells were collected after treated with PD173074 and infected with ShFGFR2 and ShCtrl lentivirus. Western blot was applied to detect the protein levels of PTEN and pPTEN.9. Statistical analysis:SPSS 17.0 software was used for processing data. Student’s t test and nonparametric test were used for statistical analysis. P values less than 0.05 were considered statistically significant.Results:1. The change of signaling pathways in damaged BMSCs and HUVECs:Western blot confirmed that the phosphorylation levels of NF-κB P65 and P38 MAPK were significantly increased in HS-5 and HUVECs after treated with ADR, IDA or Ara-C compared with controls. And the protein level of β-catenin was significantly increased in the cytoplasm and nucleus of HS-5 cells treated with ADR, IDA or Ara-C compared with controls. However, there was no significant change of mTOR phosphorylation levels.2. The effect of blocking NF-κB signaling pathways of BMSCs on the biological behaviors of AML cells and the expressions of DNA damage secreted proteins.2.1 The inhibition of NF-κB signaling by Bay11-7082:Western blot results showed that the phosphorylation level of NF-κB P65 and the protein level of IKBa were significantly reduced in HS-5 cells after exposure to Bay11-7082 for 6 hours.2.2 The effect of blocking NF-κB signaling pathways of BMSCs on the drug-sensitivity of AML cells and apoptosis.(1) The results of CCK8 and flow cytometry showed that the relative survival rate of of AML cells were significantly reduced and no significant change of apoptosis was found after cultured with conditioned medium from Bayll-7082-treated HS-5 cells compared with controls.(2) The results of CCK8 and flow cytometry showed that the relative survival rate of of AML cells were significantly reduced and the apoptosis rate was slightly increased after cultured with conditioned medium from HS-5 cells treated with Bayl 1-7082 combined with ADR compared with controls.2.3 The effect of blocking NF-κB signaling pathways of BMSCs on the expressions of DNA damage secretory proteins:Real-time RT-PCR results showed that the mRNA expressions of IL6, IL8 and Activin A were significantly reduced and the mRNA expressions of 6Ckine, FGF10, EG-VEGF and GH were significantly elevated in Bay11-7082 group compared to controls. However, there was no significant change of CYR61 mRNA expression.3. The expression of FGF10 is regulated by P-catenin pathway in BMSCs after DNA damage:Cell immunofluorescence results showed that the level of β-catenin was significantly increased in LiCl-treated HS-5 cells compared controls. Western blot determined that the protein levels of β-catenin, c-Myc, CyclinD1, CD44 and FGF10 was significantly increased in LiCl-treated HS-5 cells compared controls. Real-time RT-PCR determined that no significant change of c-Myc and CyclinD1 mRNA expressions was found while the mRNA expressions of Trib2 and FGF10 was slightly increased in HS-5 cells after treated with different concentrations of LiCl.4. Detection of FGFRs (FGFR1, FGFR2 and FGFR3) expressions.4.1 FGFRs expressions in AML cells:Real-time RT-PCR results showed that FGFRl, FGFR2 and FGFR3 mRNA expressions were higher in THP-1, U937 and Kasumi-1 cells.4.2 FGFRs expressions in the BM of AML patients:Real-time RT-PCR determined that the mRNA expressions of FGFR1 and FGFR2 were significantly increased in chemo-treated AML patients compared with controls and FGFR3 mRNA expression was significantly increased in chemo-treated AML patients compared with newly diagnosed AML patients.4.3 The effect of the conditioned medium from damaged BMSCs on the FGFRs expressions in AML cells:Real-time RT-PCR determined that the mRNA expressions of FGFR1 and FGFR2 were significantly increased in AML cells cultured with conditioned medium from IDA-treated HS-5 compared with conditioned medium from NS-treated HS-5 and RPMI 1640 medium.5. FGF10 was participated in AML drug resistance through activation of FGFR2 and downstream MAPK/STAT3 signaling pathways.5.1FGF10 activated the protein expressions of FGFR1 and FGFR2 in AML cells: Western blot and Real-time RT-PCR showed that the protein expressions of FGFRl, FGFR2 and the downstream activated molecule pFRS2α were significantly increased in THP-1 and U937 cells treated with different concentrations of FGF10 while no significant change of mRNA expressions were found.5.2FGF10 activated MAPK/STAT3 signaling pathways of AML cells:Western blot results showed that the phosphorylation levels of P38 MAPK, AKT, ERK and STAT3 were significantly increased in AML cells after treated with different concentrations of FGF10.6. Blocking FGFR2 expression in AML cells reversed AML drug resistance through inhibition of MAPK/STAT3 pathways.6.1 Blocking FGFR2 expression can enhance the drug sensitivity of AML cells:CCK8 results showed that the relative survival rate of AML cells was significantly reduced in BGJ398-treated and PD173074-treated groups compared with DMSO-treated group.6.2 Blocking FGFR2 expression can inhibit the downstream MAPK/STAT3 signaling pathways in AML cells:Western blot results showed that the protein levels of FGFR2 and pFRS2α were significantly reduced in BGJ398-treated and PD173074-treated groups compared with DMSO-treated group. Compared with DMSO-treated group, the phosphorylation levels of P38 MAPK and AKT were significantly decreased and no significant change of STAT3 phosphorylation level was found in BGJ398-treated group. Compared with DMSO-treated group, the phosphorylation levels of P38 MAPK, ERK and STAT3 were significantly decreased in PD173074-treated group.7. Down-regulation of FGFR2 expression in AML cells can reverse AML drug resistance.7.1 Detection of the FGFR2 infection efficiency:Real-time RT-PCR and Western blot determined that the mRNA and protein expressions of FGFR2 were significantly decreased in THP-1 cells infected with ShFGFR2 compared with THP-1 cells infected with ShCtrl lentivirus.7.2 Down-regulation of FGFR2 expression in AML cells can inhibit AML cells proliferation:CCK8 results showed that the proliferation rate of THP-1 cells infected with ShFGFR2 was significantly decreased compared with THP-1 cells infected with ShCtrl lentivirus.7.3 Down-regulation of FGFR2 expression in AML cells can enhance the drug-sensitivity of AML cells:CCK8 results showed that the relative survival rate of THP-1 infected with ShFGFR2 was significantly decreased compared with THP-1 cells infected with ShCtrl lentivirus after treated with different concentrations of ADR.8. Blocking FGFR2 can activate the expression of cancer suppressor gene PTEN: Western blot results showed that the phosphorylation level of PTEN was significantly increased in PD173074-treated group compared with DMSO-treated group. And compared with THP-1 cells infected with ShCtrl lentivirus, the phosphorylation level of PTEN was slightly increased in THP-1 cells infected with ShFGFR2.Conclusion:1. NF-κB and P38 MAPK signaling pathways were activated in BMSCs after DNA damage, and blockage of NF-κB signaling pathway can enhance the drug-sensitivity of AML cells and induce apoptosis, indicating that NF-κB signaling pathway plays an important role in chemotherapy-induced DNA damage to BMSCs mediated AML drug resistance.2. The expression of FGF10 is regulated by β-catenin pathway in BMSCs after DNA damage and FGF10 secreted by damaged BMSCs mediated AML drug resistance in the paracrine manner through activation of FGFR2/MAPK/STAT3 signaling pathway.3. Blockage of FGFR2 expression can reverse AML drug resistance though inhibition of MAPK/STAT3 signaling pathways, indicating that targeting receptor tyrosine kinase FGFR2/MAPK/STAT3 pathways maybe a novel strategy for overcoming AML drug resistance.Section III The aberrant expressions of Th cells in AMLBackground:In recent years, the abnormal immune function in AML patients attracted more and more attentions of scholars with the investigation of the pathogenesis of leukemia. Currently, immunologists generally agree that the dysfunction of immune system is participated in the pathogenesis of AML, which is one of the important reasons for AML recurrence and high mortality. T cell-mediated cellular immunity plays an important role in the antitumor immunity and immune regulation of organism, and the helper T cell (Th) is the important regulator and effector of the immune response. Recent studies have shown that leukemic cells suppress the host immune system with tumor progression, which is correlated with dysfunction of Th cells. However, the specific mechanisms of Th cells dysfunction in the pathogenesis of AML have not been fully elucidated.Th cells are a group of functional T cells assisting T and B lymphocyte responses. The past study of Th cells is mainly limited to the classical Thl and Th2 subtypes, and there has been reported about such Th subtypes in leukemia. Most scholars thought that Thl/Th2 ratio was decreased in AML and speculateed that there existed immune imbalance of Thl hypofunction and Th2 hyperfunction in AML. However, the relationship between Thl/Th2 and other new Th subtypes in the peripheral blood of AML patients needs further research. Th17 cells are recently identified novel CD4+ T cells that are characterized by the production of interleukin-17 (IL-17) and play an important role in the pathogenesis of chronic inflammatory, tumor and autoimmune diseases. Studies found that the proportions of Th17 cells and cytokines IL-17 were significantly increased in newly-diagnosed AML patients but obviously decreased in complete remission patients with AML. Other reports indicated that there is no obvious change of IL-17 in newly-diagnosed AML patients compared with controls. Thus, the role of Th17 cells in AML is still controversial and requires further elaboration. Th22 cells are newly identified inflammatory CD4+T cells that produce IL-22 but do not express IL-17 or IFN-γ, which has a unique gene expression and function characteristics, and play an important role in inflammation and autoimmune diseases. Our previous study showed that there...