| BackgroundAcute myeloid leukemia (AML) is malignant clonal heterogeneous disease, which has the characteristics such as being dangerous, high mortality, high frequency of recurrence. A large number of studies have shown that leukemia stem cell (LSC) is the source of drug resistance and relapse in AML. Thus eradication of LSC plays the key role to cure leukemia. So far the more generally accepted view about the LSC sources is that LSC derived from the mutations of HSC.In recent years, the treatment in AML has made great progress and the current treatment for AML is mainly chemotherapy and allogeneic hematopoietic stem cell transplantation (allo-HSCT).However, a lot of patients finally relapse. LSC which is resistant to conventional chemotherapy plays a central role in AML relapse. The residual LSC with the potential of self-renewal, proliferation and differentiation leads to the relapse. Allo-HSCT is an important way to cure AML, and alloreactive NK cells (allo-NK) plays a key role in graft anti-leukemia effect. However, the AML relapse even after allo-HSCT because of the existence of LSC, which is resistant to chemotherapy as well as allo-NK cells, remains a great challenge. So it is a critical need to study the immune attack for NK cells against LSC to ultimately curing of AML patients.As a part of innate immune system, Allo-NK cells can express activated receptors (KAR) and inhibitory receptors (KIR), which contribute to identifying normal and abnormal tissue cells and killing leukemia cells. Generally, the normal tissue cells exhibit more inhibitory signals to avoide the occurrence of autoimmune diseases. As for the tumor cells, the activated receptor KAR dominant play a leading role after losing the inhibited effect of KIR. Then NK cells are activated after activating receptor NKG2D and DNAM-1 combining with the immune activator on tumor cells’surface. NK cells may directely lysis tumor cells through secreting perforin and granzyme or kill tumor cellsthrough activating extrinsic apoptosis pathway by combining the TRAIL and FasL of NK cells with death receptors DR4/ 5 and Fas of tumor cells. Wherein, NKG2D together with its ligand MHC-like molecules (MHC class I chain related A and B, MICA/B) and UL16 binding protein family (UL 16-binding protein 1-3, ULBP1-3), constitute an important anti-tumor immune surveillance signaling pathway.The cytotoxic activity of allo-NK cells on leukemia cells are affected by the expressions of NKG2DL. Leukemia cells escape immune killing by downregulation of NKG2DL.We have found that the low expression of NKG2DL in LSC,so LSC might escape immune killing by altering the expressions of NKG2DL. Therefore, the selective upregulation of NKG2DL on LSC may increase the susceptivity to NK cells.Multiple signal transduction pathways are activated abnormally in LSC.The most important one called RAS has been found play a critical role in apoptosis resistance and escaping immune therapy. RAS is protein product of proto-oncogene ras. Cytoplasmic precursor form of RAS must be modified by farnesylation in order to locate in the inside of cell membrane, and then conducting tyrosine kinase mitogenic signals. RAS in LSC can not only activating PI3K/AKT to resist apoptosis, but also may decrease the expression level of NKG2DL to escape immune therapy. It has been proved that the low expression level of NKG2DL is related to activated ras gene and activity of DNA methyltransferase. Manumycin is being studied in the treatment of cancer based on RAS oncogene,which can inhibit farnesyl transferase enzymes.The amideside chains of manumycin are similar to farnesyl pyrophosphate isoprene group, which can competitively binding to the farnesyl transferase enzyme that can block the RAS-related mitogenic effects and induce apoptosis of tumor cells.So we assume that manumycin be related to immunomodulatory effects and explore the immune sensitizing effect of manumycin and the underlying mechanism by measuring the activity of LSC cells, the cytotoxicity of allo-NK cells against LSC, the apoptosis induced by allo-NK cells in LSC, the expressions of NKG2DL and apoptosis related protein in LSC.MethodsChapter Ⅰ Manumycin enhances the sensitivity of LSC to NK cells killing and inducing apoptosis in LSC1. LSC was separated from KGla cells using immunomagnetic beads. Flow cytometry were used to detect the purity of CD34+CD38- antigen expression on sorted cells. Allo-NK cells were isolated and purified from PBMC of healthy donors. The cytotoxicity of different concentrations of manumycin on LSC was used CCK-8.Cytotoxicity of NK cells against LSC were measured by LDH releasing assay. The apoptosis induced by NK cells in LSCs were detected by flow cytometry.2. The experimental data were analyzed using SPSS 20.0 software and represented as mean ± standard deviation (X±s). The two groups were compared using independent samples t-test analysis.The multiple groups were compared using single factor analysis of variance. To analyze at different effector to target ratio the cytotoxic of NK-92/allo-NK cells, factorial design ANOVA analysis were used. P< 0.05 was considered statistically significant.Chapter II The related mechanisms of manumycin enhancing the sensitivity of NK cell killing LSC and inducing apoptosis in LSC1. The expressions of NKG2D ligands including MICA/B and ULBP1-3 on LSCs were detected by flow cytometry.Western-blot were used to detect the key proteins of apoptosis pathway in LSC.2. The experimental data were analyzed using SPSS 20.0 software and represented as mean ± standard deviation (X±s). The NKG2DL on K562 cells and LSC were compared using independent samples t-test analysis,so did the NKG2DL on LSC before and after intervention of manumycin. P<0.05 was considered statistically significant.ResultsChapter I Manumycin enhances the sensitivity of LSC to NK cells killing and inducing apoptosis in LSC1. The isolation and identification of CD34+CD38- LSCLSC were separated from KGla cells successfully. The purity of CD34+ CD38-antigen expression on sorted cells was 99.98%.2. The morphology of allo-NK cellsAllo-NK cells were isolated from peripheral blood of healthy volunteers and began to proliferate the next day. They were smaller, circular, transparent and growth like clusters.3. The cytotoxicity of different concentrations of manumycin on LSCWe found that manumycin has cytotoxic effects on LSC in adose-dependent manner after the treatment of manumycin on LSC for 24h. LSC reached half inhibition when the concentration was 4 umol/1, namely IC50.4. Cytotoxicity of NK cells against LSCThe killing rates of the NK-92 cells to K562 cells were higher than to LSC (47.00 ± 4.31% Vs 24.08 ± 1.23%,53.58 ± 3.09%Vs 27.91 ± 2.11%,67.97 ± 3.09% Vs 34.89 ± 4.12%, respectively) at the effector-target ratios of 5:1,10:1,20:1 (F= 4.055, P<0.05). It is significantly different. Similarly, the killing rates of allo-NK cells to K562 cells were also higher than to LSC (43.38 ± 1.83%Vs 24.05 ± 3.61%, 66.80 ± 1.58%Vs 28.40 ± 3.24%,73.99 ± 4.17%Vs 35.07 ± 2.58%, respectively)at the effector-target ratios of 5:1,10:1,20:1 (F= 20.947, P<0.05).The killing rates of NK-92 cells to LSCs treated with 4 umol/1 manumycin for 24 hours were significant higher than that to LSCs without treatment(47.53 ± 3.35% Vs 24.08 ± 1.23%,62.78 ± 2.70%Vs 27.91 ± 2.11%,72.65 ± 3.38%Vs 34.89 ± 4.12%, respectively) at the effector-target ratios of 5:1,10:1,20:l,and the difference was statistically significant (F= 9.704, P0.05). The killing rates of allo-NK cells to LSCs treated with 4 umol/1 manumycin for 24 hours were also significant higher than that to LSCs without treatment^ 1.51 ± 2.94%Vs 24.05 ± 3.61%,89.47 ± 1.07%Vs 28.40 ± 3.24%,93.47 ± 0.90%Vs 35.07 ± 2.58%, respectively) at the effector-target ratios of 5:1,10:1,20:1 (F= 11.456, P0.05).5. The apoptosis induced by allo-NK cells in LSCFlow cytometry results showed that apoptosis rate of manumycin group was higher compared to the control group (8.16±0.36%Vs 1.19±0.37%), it was significantly different (P<0.05). At the effector-target ratio of 10:1, manumycin significantly enhanced the apoptosis of LSCs induced by allo-NK cells (11.38 ± 0.47%Vs 4.13 ± 0.33%,P<0.05).Chapter IIThe related mechanisms of manumycin enhancing the sensitivity of LSC to NK cell killings and inducing apoptosis in LSC1. Manumycin up-regulates the expressions of NKG2D ligands on LSCThe expressions of NKG2D ligands in LSC including MICA/B, ULBP1, ULBP2, ULBP3 were significantly lower than K562 cells and the difference was significantly different (P<0.05). This suggested that the low expressions of NK.G2D ligand may be contributed to the resistance of LSC to NK cells.The expressions of NKG2D ligands (MICA/B、ULBP1、1UJLBP2、ULBP3) on LSC treated with 4 umol/1 manumycin for 24 hours were significantly increased(8.38±0.12%Vs 0.62 ±0.26%, 12.94±3.16Vs 0.33±0.15%,9.24±0.96%Vs 0.83±0.15%,43.03±2.65%Vs 0.66±0.44%,respectively, P<0.05).2. The effects of manumycin on the key proteins of apoptosis pathway in LSCAfter the treatment of manumycin on LSC for Oh,6h,12h,18h,24h, we used Western blotting to detect proteins. The results showed that Fas gradually increased and pro-caspase8 gradually reduced.This indicated that manumycin may activate extrinsic apoptosis way. Moreover, manumycin also activated pro-caspase9 and its downstream effectors including pro-caspase3 and pro-PARP. The data suggested that manumycin might enhance the sensitivity of LSC to NK cells through activating mitochondria apoptosis pathway. In BCL-2 family, anti-apoptotic protein Bcl-xl and Bcl-2 were gradually decreased but pro-apoptotic protein Bax did not change over time.Conclusions1. Manumycin has cytotoxic effect on LSC.2. LSC resist to NK-92 cells and allo-NK cells kiliing.Manumycin can enhance the sensitivity of LSC to NK Cell-mediated killing.3. Manumycin may enhance the sensitivity of LSC to NK Cell-mediated killing through upregulating the expressions of NKG2D ligands on LSC.4. Manumycin may enhance the sensitivity of LSC to NK Cell-mediated killing through upregulating the expressions of the key molecule Fas in extrinsic apoptosis pathway as well as downregulating Bcl-xl and Bcl-2 to activate mitochondrial apoptosis pathway. |
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