| Objective:Acute myeloid leukemia(AML) is a cloned disease, which starts from malignant proliferation of immature myeloid cells of the hematopoietic system and produces an abnormal hematopoiesis. Leukemia stem cell(LSC) exists in the bone marrow(BM)microenvironment and has self-renewal capacity, which is capable of initiating and maintaining the leukemia clonal hierarchy. Most LSCs, like their normal counterparts-reside in G0, are sheltered from the traditional chemotherapies that target cycling cells and are responsible for most of the relapse in AML patients.Studies have demonstrated that AML-LSCs require interactions with supportive microenvironments(niches) to maintain their stem cell properties. Anti-CD44 antibody disrupts these interactions and eradicated LSCs, however, normal hematopoietic stem cells(HSC) are not affected by anti-CD44 antibody treatment.Our preliminary data have indicated that anti-CD44 antibody(A3D8) can reduce the adhesion of LSC but not HSC to stromal cells(SC) and osteoblast(OB), and the latter is more significant. Further, scratch assay and CCK-8 have demonstrated that A3D8 impair the migration and proliferation capacity of OB. Therefore, it’s presumed that CD44 antibody may damage the BM microenvironment. This study aimed to confirm HSC and LSC have different reactivity to the same anti-CD44 antibody and investigate the mechanisms of the CD44 ligation action to provide a theoretical basis for clinical treatment.Methods:We cultured human megakaryocytic leukemia cell line M07 e and extracted CD34+ cells from AML patients’ bone marrow and full term fetal cord blood(CB),AML CD34+CD38- and CB CD34+ cells, as representing the LSC and HSC respectively. Cell line h FOB1.19 isolated from normal adult bone marrow represented the OB in the niche. HSC/LSC and OB cells contact or transwell non-contact co-cluture system was established, and then scratch test was conducted to observe the influence of A3D8 on the migration capability of OB. Wnt/β-catenin pathway plays an important role in stem cell survival and self-renewal. Immunohistochemistrydetected expression of β-catenin in HSC/LSC and OB. Detection of A3D8 on the expression quantity of β-catenin in HSC/LSC or OB by western blot analysis. Thenβ-catenin expression quantity of OB was detected by the same method, here OB was co-cultured with HSC or LSC respectively in A3D8. Immunofluorescence was used to observe the cellular location of β-catenin in OB after treatment with A3D8.Results:1. The migration of h FOB1.19 was reduced after 24-hour treatment by A3D8,however, co-culturing with CB CD34+ cells could reverse this action. As time went on, h FOB1.19 gradually migrated to scratches gap whose width was significantly reduced. h FOB1.19 migration was not obvious in the transwell non-contact co-culture assay, which indicate that direct contact was required for the restoration of OB migration capacity. On the other hand, M07e/AML CD34+CD38- cells did not have this ability.2. The expression of β-catenin in h FOB1.19 was positive(100%) and much higher than that in M07e/AML CD34+CD38- cells and CB CD34+ cells which were negative and extremely low.3. In 5μg/ml drug concentration, h FOB1.19, CB CD34+ and M07 e cells were treated with A3D8 for 0, 24, 48 and 72 hours. A3D8 downregulated the expression quantity of β-catenin in h FOB1.19, and time-dependent effect obviously. β-catenin/GAPDH expression were(98.52 ± 6.04) %,(94.19 ± 10.05)%,(79.86 ± 13.96)% and(44.94 ± 9.40)% respectively. There were statistical differences in the groups with A3D8 treatment for 48 h and 72h(P<0.05). We were unable to observe the inhibition effect of A3D8 on CB CD34+ cells or M07 e cells.4. After co-culturing with A3D8(5μg/ml) treatment for 48 hours,β-catenin/GAPDH expression in h FOB1.19 of groups(OB, OB+A3D8, OB+CBCD34++A3D8 and OB+M07e/AMLCD34+CD38-+A3D8) were(92.40±12.74%),(77.21±9.97)%,(92.75±10.79)%,(69.31±9.11)% respectively. Co-culturing with CB CD34+ but not M07e/AML CD34+CD38- cells reversed the inhibitory effect of A3D8 and restored the h FOB1.19 down-regulation of β-catenin(P<0.05).5. In 5μg/ml drug concentration, h FOB1.19 was treated with A3D8 for 0, 24, 48 and 72 hours. The cellular location of β-catenin tended to decrease with A3D8 in atime dependent manner. It was more significant after 48 h treatment.Conclusion:1. Anti-CD44 antibody selectively eradicates LSC by interrupting the function of OB in BM microenvironment, while HSC can repair the damage without being affected by CD44 antibody.2. Ligation of CD44 by specific antibody has some inhibitory effects on the migration and proliferation capacity of OB by downregulating β-catenin expression and cellular location, and then disrupts the interactions of LSC and BM microenvironment.3. Anti-CD44 antibody can eliminate LSC but leave normal HSC unharmed by interfering Wnt/β-catenin signaling pathway. |
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