Sensitivities Of CD34~+ Early Acute Myeloid Leukemia Cells To Cytotoxicity Of Activated Immune Cells Enhanced By Plant Polyphenols

Background and ObjectiveLeukemia is a malignant disease in hematopoietic system because of expansion of colony-forming cell on some developing stage. It is the highest incidence rate that can lead the young and adult death, which occurs four-five per hundred thousand people every year in China. Recently, the incidence rate of leukemia is increased and the cure of leukemia is more difficult because of accumulative damage caused by exacerbated environment.There are leukemia stem cells with long-term self-renewal ability in leukemia that leading to occurrence of leukemia and becoming the chief source of drug resistant and relapse after treatment. Leukemia stem cells are resist to regular dose drugs and radiation damage, even to lethal dose drugs (Bu 16mg/kg and Cy 60mg/kg) and total body radiation (1100cGy) because leukemia stem cells are often lying on the rest stage(Go stage) of cell cycle. Leukemia in the body undergoes the course of immune selection and immune edition including immune surveillance, immune resistance and immune escape, therefore leukemia cells have powerful proliferation ability, especially leukemia stem cells, to escape immune surveillance through various mechanism including gene mutation. Then survival leukemia stem cells through immune escape obtain proliferation for colony and develop into leukemia finally. It is a key to clean up leukemia stem cells with drug-resistance and immune-resistance at uttermost to decrease leukemia relapse and to cure leukemia.Recently, there is a type of chemical compound with different construction, plant polyphenols, which are enriched in food and beverage consumption leading to low rate of cancer occurrence. In vivo, this compound has the ability to prevent tumor from chemicals, to inhibit tumor to induce, promote, develop and form colony, and to delay the survival of nude mice with tumor, suggesting that it can attack tumor stem cells and that it can provide a new strategy to eliminate leukemia stem cells.Therefore, in this study with CD34+ early acute myeloid leukemia KG-1a cells including leukemia stem cells as targeted cells, two kinds of plant polyphenols including resveratrol and curcumin as intervention factors, combined with immune cells activated by cytokines, according to indicators of cell proliferation, cell cycle, cell apoptosis, cytotoxicity ability and colony-forming cells and so on, the sensitivities of KG-1a cells to cytotoxicity of activated immune cells will be evaluated, the changes of the expression of immunoactivators (NKG2D ligands and DNAM1 ligands) on the surface of KG-1a cells will be detected, the role of extrinsic apoptosis pathway,including Fas pathway and TRAIL pathway, in the course of KG-1a cells to cytotoxicity of activated immune cells will be anlysesed, which will provide theoretical and experimental basis for the effective removal of leukemia stem cells and new strategy for curing leukemia.MethodsChapterⅠEffects of plant polyphenols on biological properties of CD34+ early acute myeloid leukemia cellsCells strain including leukemia stem cells was selected as targeted cells for following study by the ways of observation through inverted phase contrast microscope and cells in smear stained with Wright-Giemsa dye, counting cells rejecting to dye with trypan blue, expressions of CD34CD38 through flow cytometry and colony-forming ratio through semisolid culture. The concentrations of plant polyphenols (resveratrol and curcumin) for 50% survival ratio were selected. Different effects of resveratrol and curcumin on biological properties of CD34+early acute myeloid leukemia cells were compared through changes of morphology, apoptosis, colony-forming ratio and caspase8 activity.ChapterⅡEffects of plant polyphenols on cytotoxicity of activated immune cells to CD34+ early acute myeloid leukemia cellsMononuclear cells from peripheral blood were separated through density-gradient centrifugation, which were activated through addition with cytokines. Then activated immune cells were identified through morphology of living cells, cells in smear stained with Wright-Giemsa dye, flow cytometry and cytotoxicity to K562 cells. After resveratrol and curcumin cultured with KG-1a cells for 24h, living KG-1a cells cultured with activated immune cells at different ratio, then changes of morphology were observed through inverted phase contrast microscope, cytotoxicity to KG-1a cells were measured by LDH releasing assay, changes of colony-forming ratio were analyzed through semisolid culture method.ChapterⅢRegulation of plant polyphenols to CD34+ early acute myeloid leukemia cells for immunoactivators and death receptors in extrinsic apoptosis pathwayAfter resveratrol and curcumin cultured with KG-1a cells for 24h, expression of immunoactivators and death receptors in extrinsic apoptosis pathway on the surface of living KG-1a cells were detected through flow cytometry, including NKG2D ligands, DNAM1 ligands, Fas and Fas ligand, TRAIL receptors. Then added with TRAIL, proliferation ratios of KG-1a cells were measured by XTT proliferation and cytotoxicity assay kit, colony-forming ratios were analyzed through semisolid culture method, cell cycle and apoptosis were detected by flow cytometry, caspase8 activity were analyzed by spectrophotometric method, apoptosis-relating gene mRNA were measured by real-time PCR.ChapterⅣEffects of CD34+ early acute myeloid eukemia cells treated with plant polyphenols to activated immune cellsAfter resveratrol and curcumin cultured with KG-1a cells for 24h, KG-1a cells for living cultured with activated immune cells at effector-target ratio for 10:1 for 4h, expression of NKG2D and DNAM1, Fas and Fas ligand, TRAIL receptors on the surface, apoptosis of activated immune cells were detected through flow cytometry, caspase8 activity in activated immune cells were measured by spectrophotometric method.Statistical analysisAnalyses were performed using SPSS 13.0 software package. Data were represented as the mean±standard deviation (x±s). Comparisons of means among groups were performed using one-way analysis of variance (ANOVA). If variances were not homogenous among groups, correct method of F test by Welch was performed. If variances were homogenous among groups, the LSD method was used for multiple comparisons; otherwise Dunnett's T3 method was used. Pairwise comparison was performed using compared samples t-test. Factorial design analysis of variance was performed using repeated measures. P<0.05 was considered to be statistically significant.ResultsChapterⅠEffects of plant polyphenols on biological properties of CD34+ early acute myeloid leukemia cells1.1 Characteristics of CD34+ early acute myeloid leukemia cellsKG-1a cells and HL-60 cells are bright, round and well in reflected light, with homogenous size and clear edge under inverted phase contrast microscope. KG-1a cells in smear stained with Wright-Giemsa, have characteristics of increasing rate of cytoplasm and nucleus, vacuoles intracytoplasmia with no particles, bigger size nuclear with soften chromatin and prominent nucleoli, while HL-60 cells in smear stained with Wright-Giemsa, have characteristics of decreasing rate of cytoplasm and nucleus, many particles intracytoplasmia, smaller size nuclear with compact chromatin and rare nucleoli. The doubling logarithmic-growth time of KG-la cells is 24.24h, longer than 15.83h of HL-60 cells. KG-la cells can express CD34 antigen and some absent in CD38 antigen, and HL-60 cells can not express CD34 and CD38 antigens.14-day colonies from KG-la cells are less than from HL-60cells and 21-day colonies from KG-la cells are more than from HL-60 cells, suggesting that KG-la cells are on the earlier stage of differentiation than HL-60 cells and include many leukemia stem cells.1.2 Effects of plant polyphenols on biological properties of CD34+ early acute myeloid leukemia cells1.2.1 Effects of resveratrol and curcumin on proliferation of KG-la cellResveratrol and curcumin both could inhibit the proliferation of KG-la cells with a dose and time dependent manner. Resveratrol could inhibit the proliferation of peripheral blood mononuclear cells (PB-MNC) with some degree, but 25-100umol/L resveratrol had no effect on proliferation of PB-MNC. And 50% inhibition concentration of resveratrol to KG-la cells at 24h and 48h both are 25umol/L, which had no effect on PB-MNC. Therefore,25umol/L resveratrol was selected as intervention for following study. Curcumin could inhibit the proliferation of PB-MNC, but 12.5umol/L curcumin had no effect on proliferation of PB-MNC. And 50% inhibition concentration of curcumin to KG-la cells at 24h is 12.5umol/L. Therefore, 12.5umol/L curcumin were selected as intervention for following study.1.2.2 Effects of resveratrol and curcumin on morphology of KG-la cells Through inverted phase contrast microscope, KG-la cells as control were bright and round with clear and smooth edge and with no debris, while KG-la cells with resveratrol and curcumin for 24h had irregulated size, many granules intracytoplamsia and some debris. Through fluorescent microscope with DAPI stained, KG-la cell nucleus as control had homogenous stain, regulated size and complete edge, while KG-1a cell nucleus reated with resveratrol and curcumin for 24h had irregulated size, inhomogenous stain and incomplete edge, even cauliflower-like and piecemeal necrosis.1.2.3 Effects of resveratrol and curcumin on colony-forming cells of KG-la cellsThrough semisolid culture with methylcellulose, after 14 days culture, KG-la cells could form colonies including more than 50 cells. Then the number and the size of colony were increasing with time to the maximum at 21 days. Finally, cells in the colonies would dye and the colonies would loose. Contrast to control, resveratrol and curcumin could inhibit the number of colony at 14 days and 21 days, suggesting that resveratrol and curcumin could inhibit the prolirferation of conoly-forming cells.1.2.4 Effects of resveratrol and curcumin on cell cycle of KG-1a cellsChanges of cell cycle would be detected by flow cytometry. Contrast to control, KG-la cells treated with resveratrol had an increase on G0/G1 phase and a decrease in S and G2/M phase, suggesting that resveratrol could inhibit KG-1a cells to rest on G0/G1 phase. There were no changes in cell cy;le of KG-1a cells treated with curcumin.1.2.5 Effects of resveratrol and curcumin on CD34CD38 expression of KG-la cellsChanges of CD34CD38 expression of KG-la cells would be detected by flow cytometry. Resveratrol could decrease the number of KG-1a cells on late stage and make them rest on early stage, while curcumin could decrease the number of KG-1a cells on early stage and induce them to different to mature. 1.2.6 Effects of resveratrol and curcumin on apoptosis of KG-la cellsResveratrol and curcuim both could induce KG-la cells to apoptosis through flow cytometry and fluorescent microscope methods.1.2.7 Effects of resveratrol and curcumin on caspase8 activity of KG-la cellsChanges of caspase8 activity would be measured by spectrophotometric method. Contrast to control, caspase8 activity of KG-la cells treated with reaveratrol and curcumin for 12h increased, then decreased as time prolonging. ChapterⅡEffects of plant polyphenols on cytotoxicity of activated immune cells to CD34+ early acute myeloid leukemia cells2.1 Characteristics of activated immune cells2.1.1 Morphology of activated immune cellsCells were small, round, bright, suspended and tighted with colony-like form and clear edge under inverted phase contrast microscope, while cells, in smear stained with Wright-Giemsa, were small, round, blue and no particles intracytoplasmia, tight chromatin in the nucleoli, characteristic of lymphocyte.2.1.2 Cell cycle and apoptosis of activated immune cellsActivated immune cells with cytokines IL-2 and IL-15 could not be apoptosis spontaneously. Most of cells on the stage of G0/G1 were difficult to be activated by surroundings.2.1.3 Activating receptors expression of activated immune cellsActivating receptors NKG2D expression of activated immune cells was (51.27±7.41)% and DNAM1 was (47.10±3.21)%, suggesting that cytokines IL-2 and IL-15 could activate PB-MNC to express activating receptors.2.1.4 Intrinsic apoptosis pathway Fas/FasL and TRAIL expression of activated immune cellsIntrinsic apoptosis pathway TRAIL expression of activated immune cells was (44.17±7.22)%, Fas was (89.03±7.14)% and Fas Ligand was (19.20±4.70)%.2.1.5 Cytotoxicity of activated immune cells to K562 cellsCytotoxicity of activated immune cells to K552 cells was measured by LDH assay. When effect to target was 10:1, the cytotoxicity was (61.67±16.47)%, while effect to target was 20:1, the cytotoxicity was (85.56±8.88)%, suggesting that activated immune cells had high cytotoxicity withou:MHC-Ⅰmolecule.2.2 Effects of plant polyphenols on cytotoxicity of activated immune cells to CD34+ early acute myeloid leukemia cells2.2.1 Effects of resveratrol and curcumin on cytotoxicity of activated immune cells to KG-la cells by LDH assayKG-la cells treated with resveratrol and curcunin were cultured with activated immune cells, they would be observed under inverted phase contrast microscope that activated immune cells were smaller and surrounded with bigger KG-la cells. Through LDH assay, when effect to target was 10:1, resveratrol and curcumin could improve the cytotoxicity of activated immune cells to KG-1a cells; when effect to target was 20:1, resveratrol, not curcumin, could improve the cytotoxicity of activated immune cells to KG-la cells.2.2.2 Effects of resveratrol and curcumin on cytotoxicity of activated immune cells to colony-forming cells from KG-la cellsEffects of resveratrol and curcumin on cytotoxicity of activated immune cells to colony-forming cells from KG-la cells were analyzed by the way of semisolid culture. Activated immune cells could inhibit 21-day, but not 14-day, colony-forming cells from KG-la cells, and resveratrol could enhance the sensitivities of cytotoxicity of activated immune cells to 21-day, but not 14-day, colony-forming cells from KG-la cells, while curcumin could enhance the sensitivities of cytotoxicity of activated immune cells to 14-day and 21-day colony-forming cells from KG-la cells. ChapterⅢRegulation of plant polyphenols to CD34+ early acute myeloid leukemia cells for immunoactivators and death receptors in extrinsic apoptosis pathway3.1 Effects of resveratrol and curcumin on expression of MICA, MICB, ULBP1, ULBP2 and ULBP3 as NKG2D ligands on the surface of KG-la cellsEffect of resveratrol and curcumin on MICA, MICB, ULBP1, ULBP2 and ULBP3 expression on the surface of KG-la cells was detected by flow cytometry. The results were that curcumin could improve the expression of MICA and MICB, while resveratrol could improve the expression of ULBP1, ULBP2 and ULBP3 on the surface of KG-la cells.3.2 Effects of resveratrol and curcumin on expression of CD112 and CD155 as NKG2D ligands on the surface of KG-la cellsEffect of resveratrol and curcumin on CD112 and CD155 expression on the surface of KG-la cells was detected by flow cytometry. The results were that resveratrol could decrease the expression of CD112, while curcumin could decrease the expression of CD112 and CD155.3.3 Effects of resveratrol and curcimin on expression of Fas and Fas ligand on the surface of KG-la cellsEffect of resveratrol and curcumin on Fas and Fas ligand expression on the surface of KG-la cells was detected by flow cytometry. The results were that resveratrol had no effect on the expression of Fas and Fas ligand, while curcumin could decrease the expression of Fas ligand.3.4 Effects of resveratrol and curcimin on TRAIL pathway of KG-la cells3.4.1 Effects of resveratrol and curcimin on expression of TRAIL, death receptors DR4/5 and decoy receptors DcR1/2 on the surface of KG-la cellsEffect of resveratrol and curcumin on TRAIL, death receptors DR4/5 and decoy receptors DcR1/2 expression on the surface of KG-1a cells was detected by flow cytometry. The results were that resveratrol could increase the expression of death receptor DR5 and decrease the expression of decoy receptor DcR1, while curcumin could decrease the expression of decoy receptor DcR1 and had no effect on the expression of death receptor DR4/5.3.4.2 Cytotoxicity of TRAIL combined with resveratrol and curcumin to KG-la cellsEffects of various concentration TRAIL on proliferation of KG-la cells with 25 umol/L resveratrol and 12.5 umol/L curcumin wen; analyzed by XTT proliferation and cytotoxicity assay kit. The results were that resveratrol could improve the cytotoxicity of TRAIL to KG-la cells with no dose-dependent manner, while curcumin had no effect on the cytotoxicity of TPAIL to KG-la cells. Therefore, 10ng/ml TRAIL was selected as intervention for following study.3.4.3 Effects of TRAIL combined with resveratrol and curcimin on colony-forming ratio of KG-1a cellsEffects of TRAIL on colony-forming ratio of KG-la cells treated with each resveratrol and curcumin were analyzed through semisolid culture method. The results were that TRAIL had no effect on 14-day and 21-day colony-forming ratio. Resveratrol could increase the inhibition of TRAIL to 21-day colony-forming ratio and had no effect on the inhibition of TRAIL to 14-day colony-forming ratio. Curcumin had no effect on the inhibition of TRAIL to 21-day colony-forming ratio and increase TRAIL to 14-day colony-forming ratio.3.4.4 Effects of TRAIL combined with resveratrol and curcimin on cell cycle of KG-la cellsEffects of TRAIL on cell cycle of KG-la cells treated with and without resveratrol were detected through flow cytometry. The results were that there were no changes of cell cycle in KG-la group and KG-la/RES group treated with TRAIL contrast to no TRAIL, suggesting that TRAIL had no effect on cell cycle of KG-la cells.3.4.5 Effects of TRAIL combined with resveratrol and curcimin on apoptosis of KG-la cellsEffects of TRAIL on apoptosis of KG-la cells treated with and without resveratrol were detected through flow cytometry. The results were that resveratrol could promote TRAIL to induce KG-la cells to late apoptosis and necrosis.3.4.6 Effects of TRAIL combined with resveratrol and curcimin on caspase8 activity of KG-la cellsEffects of TRAIL on caspase8 activity of KG-la cells treated with and without resveratrol were detected through spectrophotometric assay. The results were that TRAIL couldn't up-regulate caspase8 activity and resveratrol could not force TRAIL to up-regulate caspase8 activity of KG-1a cells.3.4.7 Effects of TRAIL combined with resveratrol and curcimin on bcl-2 and bcl-xl gene of KG-la cellsEffects of TRAIL on bcl-2 and bcl-xl genes of KG-la cells treated with and without resveratrol were analyzed through real-time RT-PCR. The results were that TRAIL could down-regulate the anti-apoptosis genes of KG-la cells including bcl-2 and bcl-xl, resveratrol could increase the down-regulation of TRAIL to bcl-2 gene, not bcl-xl gene, of KG-1a cells.ChapterⅣEffects of CD34+ early acute myeloid leukemia cells treated with plant polyphenols to activated immune cells4.1 Effects of KG-la cells treated with resveratrol and curcumin on expression of activating receptors on the surface of activated immune cellsEffects of KG-la cells treated with resveratrol and curcumin on expression of activate receptors on the surface of activated immune cells were detected through flow cytometry. The results were that activated immune cells cultured with KG-la cells for 4h, had no changes in the expression of NKG2D and DNAM1. KG-la cells treated with resveratrol and curcumin could decrease the expression of DNAM1 on the surface of activated immune cells and had no effects on the expression of NKG2D.4.2 Effects of KG-la cells treated with resveratrol and curcumin on expression of Fas and Fas ligand on the surface of activated immune cellsEffects of KG-la cells treated with resveratro and curcumin on expression of Fas and Fas ligand on the surface of activated immune cells were detected through flow cytometry. The results were that activated immune cells cultured with KG-la cells for 4h, had no changes in the expression of Fas and Fas ligand. KG-la cells treated with resveratrol and curcumin could decrease the expression of Fas and Fas ligand on the surface of activated immune cells.4.3 Effects of KG-la cells treated with resveratrol and curcumin on expression of TRAIL and receptors on the surface of activated immune cellsEffects of KG-la cells treated with resveratrol and curcumin on expression of TRAIL receptors on the surface of activated immune cells were detected through flow cytometry. The results were that activated immune c;lls cultured with KG-la cells for 4h, could decrease the TRAIL death receptors DR4/5 expression and increase the TRAIL decoy receptor DcRl but DcR2 expression. KG-la cells treated with resveratrol and curcumin could decrease the expression of TRAIL, death receptors DR4/5 and decoy receptors DcRl/2 on the surface of activated immune cells.4.4 Effects of KG-la cells treated with resveratrol and curcumin on apoptosis of activated immune cellsEffects of KG-la cells treated with resveratrcl and curcumin on apoptosis of activated immune cells were detected through flow cytometry. The results were that apoptosis rate of activated immune cells cultured with KG-la cells was less increasing than those of cultured with KG-1a/RES cells and KG-1a/CUR cells.4.5 Effects of KG-1a cells treated with resveratrol and curcumin on expression of caspase8 activity of activated immune cellsEffects of KG-1a cells treated with resveratrol and curcumin on caspase8 activity of activated immune cells were measured by spectrophotometric method. The results were that activated immune cells cultured with KG-1a cells for 4h, contrast to 0h, could increase caspase8 activity, while KG-1a cells treated with resveratrol and curcumin had no effect on caspase8 activity of KG-1a cells.Conclusions1. Resveratrol and curcumin could inhibit the proliferation of CD34+ early acute myeloid leukemia KG-1a cells including leukemia stem cells and could inhibit them to form colony at 14 days and 21 days, suggesting that resveratrol and curcumin had roles on leukemia stem cells.2. Resveratrol and curcumin both induced KG-1a cells to apoptosis.3. Resveratrol could increase expression of ULBP1-3 on the surface of KG-1a cells and curcumin could increase expression of MICA/B on the surface of KG-1a cells, resulting to activate the killing ability of immune cells.4. Resveratrol could up-regulate the expression of DR5 on the surface of KG-1a cells, down-regulate the expression of DcR1 and bcl-2 gene and improve caspase8 activity of KG-1a cells, resulting to increase the sensitivities of KG-1a cells to cytotoxicity of immune cells.5. Resveratrol and curcumin could prevent KG-1a cells from inducing activated immune cells to apoptosis through extrintic apoptosis pathway.