反式桂皮醛对髓细胞白血病细胞体外效应的研究
摘要
第一部分反式桂皮醛对急性髓细胞白血病细胞增殖和凋亡的影响
目的:研究反式桂皮醛(trans-cinnamaldehyde,TCA)对急性髓细胞白血病(acute myeloid leukemia,AML)细胞增殖和凋亡的影响。
方法:用不同浓度的TCA和/或阿糖胞苷(β-D-Arabinofuranosyl cytosine,AraC)处理AML细胞株HL60或AML初治患者及健康人骨髓单个核细胞( bone marrow mononuclear cell,BMMNC)。CCK-8法测定HL60细胞增殖活性,流式细胞术检测HL60细胞周期变化, Annexin V/PI双标法检测HL60细胞和BMMNC凋亡,CD45/CD34/Annexin V三标法检测BMMNC CD34+细胞凋亡。Western blot检测TCA处理后HL60细胞c-myc基因表达。激光共聚焦术检测TCA处理后HL60细胞NF-κB活性。甲基纤维素法检测TCA处理后BMMNC克隆形成。
结果:TCA呈时间和剂量依赖性影响HL60细胞增殖。低浓度(10μM,20μM)TCA作用24 h后,促进HL60细胞增殖,和阴性对照组相比,有显著差异(P<0.05)。而较高浓度TCA对HL60细胞的增殖表现出抑制效应,并随浓度增加抑制作用明显增强。各浓度TCA作用48 h和72 h均表现出对HL60细胞增殖的抑制效应。TCA作用HL60细胞24 h,48 h和72 h的IC_(50)分别为78.40μM,58.13μM和50.31μM。高浓度TCA(≥80μM)可诱导>10%细胞发生凋亡,增加TCA的作用浓度使HL60细胞凋亡率进一步增加。TCA浓度≤60μM时作用24 h后,HL60细胞凋亡率<5%。在中浓度(40μM,60μM)TCA作用下,随作用时间延长, HL60细胞凋亡率也逐渐增加。低浓度(10μM,20μM)TCA始终不能显著增加凋亡率。中浓度TCA作用24 h后,HL60细胞明显阻滞于G2 /M。低浓度TCA作用24 h对细胞周期无明显影响,作用72 h后,细胞周期阻滞于G0/G1期。HL60细胞高表达c-Myc蛋白,TCA呈时间和剂量依赖性显著抑制c-Myc蛋白的表达。100μM TCA作用4 h后,HL60细胞NF-κB失活。TCA呈剂量依赖性诱导AML BMMNC和CD34+细胞凋亡,TCA对正常BMMNC和CD34+细胞的细胞毒作用很小,差异有显著性(P<0.05)。TCA非谱系特异性抑制AML原代细胞克隆形成。TCA协同AraC对HL60细胞、AML原代细胞及AML CD34+细胞的细胞毒性作用。
结论: TCA通过使NF-κB失活,下调原癌基因c-myc的表达,使HL60细胞周期阻滞和诱导细胞凋亡,发挥抗白血病效应。TCA对AML BMMNC和CD34+细胞也产生明显的凋亡诱导效应,并能协同AraC发挥抗白血病效应,对正常BMMNC的细胞毒作用很小。
第二部分反式桂皮醛对慢性髓细胞白血病细胞增殖和凋亡的影响
目的:研究反式桂皮醛(trans-cinnamaldehyde,TCA)对慢性髓细胞白血病(chronic myeloid leukemia,CML)细胞增殖和凋亡的影响。
方法:用不同浓度的TCA处理CML细胞株K562和初治CML患者及健康人骨髓单个核细胞(bone marrow mononuclear cell,BMMNC)。CCK-8法测定K562细胞增殖活性,流式细胞术检测K562细胞周期变化、K562细胞和BMMNC凋亡及K562细胞膜电位和Fas抗原表达。Western blot检测K562细胞c-myc基因表达和Crkl蛋白磷酸化水平。Real time PCR检测K562细胞Bcr-Abl mRNA表达。甲基纤维素法检测CML BMMNC克隆形成。
结果:TCA对K562的增殖作用的影响呈时间和剂量依赖性。低浓度(30μM)TCA作用24 h和48 h促进K562细胞增殖,和阴性对照组相比,有显著差异(P<0.01);作用72 h,抑制K562细胞增殖(P<0.01)。较高浓度TCA对K562细胞的增殖始终表现出抑制效应,并随浓度增加抑制作用明显增强。TCA处理K562细胞24 h的IC50是157.42μM。低浓度(30μM) TCA始终不能诱导K562细胞凋亡,中浓度(60μM)TCA作用24 h,仅极少部分细胞产生凋亡,与对照组细胞相比,差异无统计学意义(P>0.05)。高浓度TCA(≥90μM)作用24 h后可诱导>10%细胞发生凋亡,增加TCA浓度和作用时间可诱导更多细胞凋亡。60μM TCA作用24 h使K562细胞明显阻滞于G2 / M期,而30μM TCA则轻微增加G2 /M期细胞的比例(P>0.05)。K562细胞高表达c-Myc蛋白和磷酸化Crkl,TCA呈时间和剂量依赖性明显抑制c-Myc蛋白的表达和Crkl磷酸化水平。TCA呈时间和剂量依赖性下调线粒体跨膜电位和上调K562细胞的Fas表达。TCA呈剂量依赖性抑制K562细胞的Bcr-Abl转录水平。TCA呈剂量依赖性诱导CML BMMNC凋亡,对正常BMMNC的细胞毒作用很小。TCA非谱系特异性抑制CML BMMNC克隆形成。
结论:低浓度TCA(≤30μM)早期可能促进K562细胞增殖,而后期可诱导细胞周期阻滞,从而抑制K562细胞生长。中浓度TCA(60μM)早期阻滞细胞周期于G2 /M期,而随作用时间延长,诱导细胞出现凋亡。高浓度TCA(≥90μM)则通过诱导细胞凋亡,从而抑制K562细胞的生长,凋亡率呈时间和剂量依赖性。两条主要的凋亡通路,即线粒体介导和Fas介导的凋亡通路,都参与了TCA诱导的凋亡。TCA可能通过抑制Bcr-Abl转录,从而减少Bcr-Abl蛋白产物,削弱酪氨酸激酶效应,下调c-myc癌基因的蛋白表达,导致CML细胞凋亡、抑制CML细胞增殖。以上提示TCA具有明显的抗慢性髓细胞白血病效应,对正常骨髓单个核细胞的细胞毒作用很小,是一个很有应用前景的药。
第三部分反式桂皮醛诱导髓细胞白血病细胞分化
目的:本部分旨在研究反式桂皮醛(trans-cinnamaldehyde,TCA)对髓细胞白血病细胞分化的影响及机制。
方法:用TCA和/或反式维甲酸(all-trans-Retinoic acid,ATRA)处理髓细胞白血病细胞株HL60细胞及K562细胞。相差显微镜和Wright’s-Gimsa染色观察细胞形态变化。流式细胞术检测细胞分化抗原表达、细胞周期和凋亡。Western blot检测HL60细胞c-myc基因和p27基因表达、K562细胞c-myc基因表达和Crkl蛋白磷酸化水平。激光共聚焦术检测HL60细胞p16基因和Cdc6基因表达变化。
结果: TCA(≤60μM)诱导HL60细胞向成熟粒细胞分化,细胞表面分化抗原CD11b表达增加。TCA(≤60μM)诱导K562细胞向单核-巨噬细胞分化,细胞表面分化抗原CD14和CD11b表达均增加。低浓度(20μM)TCA作用72 h使HL60细胞阻滞于G0/G1期,不诱导明显细胞凋亡;中浓度(60μM)TCA作用24 h,HL60细胞G2/M期比例明显增加,G0/ G1期细胞比例明显下降,持续作用48 h时,HL60出现G2/M细胞比例降低,出现明显凋亡。中浓度(60μM)TCA作用于K562细胞24 h后,G2/M期细胞比例明显增加,持续作用48 h后,G2/M期细胞比例进一步增加,作用72 h时,K562细胞G2/M期比例下降,出现明显凋亡。TCA诱导HL60细胞分化伴原癌基因c-myc表达下降,细胞周期相关蛋白p27表达增强。TCA诱导K562细胞分化伴c-myc基因表达下调,Crkl蛋白磷酸化水平下降。TCA诱导HL60细胞分化还伴p16蛋白表达增加,向核内移位,而Cdc6蛋白表达下降,向核外迁移。TCA可协同增强ARTA对HL60细胞的诱导分化效应。
结论: TCA(≤60μM)可诱导髓细胞白血病细胞分化,但不同种类的细胞对TCA的反应有差异性,而且同种白血病细胞对不同浓度的TCA的反应不同。TCA诱导HL60白血病细胞分化与c-myc蛋白水平下降和p27蛋白水平上调有关,p16基因和Cdc6基因参与TCA诱导的HL60细胞分化过程。TCA可增强ATRA对HL60细胞的诱导分化效应。TCA诱导K562细胞的分化与c-myc和Crkl蛋白磷酸化水平下降有关。
第四部分反式桂皮醛对急性髓细胞白血病细胞粘附和迁移的影响
目的:本部分旨在研究反式桂皮醛(trans-cinnamaldehyde,TCA)对急性髓细胞白血病(acute myeloid leukemia,AML)细胞粘附和迁移的影响。
方法:TCA处理AML细胞株HL60细胞,流式检测细胞表面CXCR4的表达。Transwell法检测不同浓度TCA对重组基质细胞衍生因子-1α(stromal cell derived factor-1α,SDF-1α)诱导的HL60迁移和浸润的影响。羧基荧光素二乙酸盐琥珀酰亚胺酯(carboxyfluorescein succinimidyl ester,CFSE)标记HL60细胞,共聚焦显微镜和流式细胞术检测HL60细胞染色情况。CFSE标记的HL60细胞与AML骨髓基质细胞(bone marrow stromal cell, BMSC)共培养,用共聚焦检测不同浓度TCA处理后HL60细胞与AML BMSC的粘附情况。ELISA法检测TCA对AML BMSC分泌SDF-1α的影响。相差显微镜观察TCA处理后AML BMSC细胞形态变化,流式细胞术检测AML BMSC细胞凋亡。
结果:TCA作用后,HL细胞CXCR4表达的平均荧光强度(mean fluorescence intensity,MFI)呈时间和剂量依赖性明显下降(P值均<0.05)。TCA呈剂量依赖性抑制HL60趋向重组人SDF-1α的迁移和浸润。CFSE标记后培养过夜的HL60细胞发出很强的绿色强荧光,在细胞核和细胞质内均有分布,而未标记的HL60细胞和BMSC未见荧光。TCA作用6 h后呈剂量依赖性抑制HL60细胞与AML BMSC间的粘附。TCA呈剂量依赖性抑制AML BMSC分泌SDF-1α。TCA作用24 h,AML BMSC细胞间隙增大。TCA呈现时间和剂量依赖性诱导AML BMSC凋亡。
结论:TCA除可直接抑制白血病细胞的增殖、诱导白血病细胞凋亡和分化外,还可能通过下调白血病细胞CXCR4表达、抑制骨髓基质细胞SDF-1分泌和诱导骨髓基质细胞凋亡、抑制白血病细胞对骨髓基质细胞的趋化,使白血病细胞一方面失去基质层的支持,另一方面易于发生凋亡,因此有可能削弱骨髓基质细胞对白血病细胞潜在的保护作用,有利于清除髓内白血病细胞。
第五部分反式桂皮醛调节髓细胞白血病细胞mel18基因表达
目的:本研究旨在检测mel18基因在急性髓细胞白血病(acute myeloid leukemia,AML)的表达,研究mel18基因对AML细胞的增殖、凋亡和周期的影响,探讨反式桂皮醛(trans-cinnamaldehyde,TCA)对AML细胞mel18基因的调控。
方法:运用逆转录聚合酶链式反应(reverse transcription polymerase chain reaction, RT-PCR)检测白血病细胞株mel18 mRNA表达,生物素-生物素-过氧化物酶(avidin-biotin-peroxidase complex,ABC)法免疫组织化学(immunohistochemistry stain,IHC)染色分析髓细胞白血病细胞株、初治AML患者骨髓单个核细胞和健康人外周血及骨髓单个核细胞的Mel18蛋白表达。运用十四酰佛波乙酸酯(phorbol-12-myristate-13-acetaet,PMA)诱导HL60细胞分化,流式细胞术、免疫组化法和激光共聚焦术检测诱导分化细胞的Mel18蛋白表达变化。通过基因重组和分子克隆技术构建pLenti6/V5-mel18真核表达载体并鉴定,应用脂质体(Lipofectamine2000?)转染,将pLenti6/V5-mel18和pLenti6/V5-LacZ(阴性对照)导入HL60和U937细胞。RT- PCR和流式细胞术检测pLenti6/V5-mel18质粒转染细胞中mel8基因的表达。用CCK-8法测定细胞增殖活性,流式细胞术检测细胞凋亡和细胞周期。TCA诱导K562细胞和HL60细胞分化,流式细胞术和激光共聚焦术检测Mel18蛋白表达。
结果:mel18 mRNA在K562细胞强表达,HL60细胞弱表达,U937细胞无表达。Mel18蛋白主要表达在细胞质中,在K562细胞和正常外周血中高表达,HL60和正常骨髓单个核细胞中低表达,而U937细胞不表达Mel18蛋白。Mel18蛋白在AML骨髓中缺失率为58.8 %,正常对照骨髓单个核细胞中未检测到Mel18蛋白缺失。HL60细胞被PMA诱导分化后Mel18蛋白表达增加,亚细胞定位不变。重组质粒pLenti6/V5-mel18经过PCR检测和公司测序证实构建成功。pLenti6/V5-mel18质粒转染24 h后, U937细胞和HL60细胞的mel18基因表达增加,转染pLenti6/V5-mel18的U937细胞和HL60细胞增殖受抑,细胞出现凋亡,G0/G1期细胞比例增加。TCA诱导HL60细胞和K562细胞分化伴Mel18蛋白表达增加,Mel18亚细胞定位不发生改变。
结论: mel18基因在AML中表达降低,mel18基因的表达与分化相关。mel18在AML中可能发挥抑癌基因的作用,增强mel18基因的表达可抑制AML细胞株增殖,诱导细胞凋亡,使细胞周期阻滞。TCA诱导白血病细胞分化可增加mel18基因的表达。
PART I Effects of Trans-cinnamaldehyde on the Proliferation and Apoptosis of Acute Myeloid Leukemia Cells
Objective To study the effects of trans-cinnamaldehyde (TCA) on the proliferation and apoptosis of acute myeloid leukemia (AML) cells.
Methods HL60 AML cells and bone marrow mononuclear cells(BMMNC)from patients with de novo AML and healthy donors were treated by various concentrations of TCA and / orβ-D-Arabinofuranosyl cytosine (AraC). The proliferation of HL60 cells were detected by CCK-8 assay. Flow cytometry was empolyed to detect the cell cycle disribution of HL60 cells. The apoptosis of HL60 cells and BMMNC was measured by Annexin V/PI double labeled, and the apoptosis of BMMNC CD34+ cells were examined by triple labled with CD45/CD34/Annexin V. Western blot assay was employed to quantify the expression of c-myc gene in HL60 cells treated by TCA.The activity of NF-κB was investigated by confocal. Furthermore, the colony formation capability of BMMNC treated by TCA were determined by cytokines-riched methylcellulose culture assay.
Results TCA affected the proliferation of HL60 cells in a time- and dose-dependent fashion. Low concentrations of TCA (10μM, 20μM) promoted the growth of HL60 cells at 24 h, different from untreated control significantly (P<0.05). Higher concentrations of TCA inhibited proliferation of HL60 cells. TCA administrated at any concentraion halted proliferation of HL60 cells at 48 h and 72 h. The values of IC_(50) of TCA on HL60 cells were 78.40μM, 58.13μM and 50.31μM at 24 h, 48 h and 72 h, respectively. High concentrations of TCA (≥80μM) induced more than 10% cells to undergo apoptosis. More apoptosis could be seen in HL60 cells treated by increased concentrations of TCA. Less than 5% HL60 cells underwent apoptosis when treated by TCA with the concentration no more than 60μM for 24 h. The apoptosis of HL60 cells treated by middle concentrations of TCA (40μM, 60μM) increased when the treated time was prolonged. Low concentrations of TCA (10μM, 20μM) could not induce significant apoptosis of HL60 cells at any time. HL60 cells were arrested at G2 /M phase after treated by middle concentraions of TCA for 24 h. Low concentraions of TCA did not affect the cell cycle of HL60 cells at 24 h but accumulated HL60 cell at G2 /M phase at 72 h. c-Myc protein was highly expressed in HL60 cells and TCA inhibited the expression of c-Myc in HL60 cells in a time- and dose- dependent manner. The activity of NF-κB in HL60 cells was inhibited by treatment of 100μM TCA for 4 h. TCA induced apoptosis of AML BMMNC and AML CD34+ cells time- and dose-dependently.The cytotoxicity of TCA on normal BMMNC was slight. TCA inhibited colony formation of AML BMMNC. Furthermore, TCA synergized the cytotoxicity of AraC on HL60 cells, AML BMMNC and AML BMMNC CD34+ cells as well.
Conclusion TCA exhibited anti-leukemia charateristic by inhibiting cell cycle progression, inducing apoptosis, deactivating NF-κB and repressing the expression of c-myc gene. TCA leaded AML BMMNC cells and AML CD34+ cells to significantly apoptosis with little cytotoxicity on normal BMMNC. Moreover, TCA synergized the anti-leukemia effect of AraC.
PARTⅡEffects of Trans-cinnamaldehyde on the Proliferation and Apoptosis of Chronic Myeloid Leukemia Cells
Objective To study the effects of trans-cinnamaldehyde (TCA) on the proliferation and apoptosis of chronic myeloid leukemia (CML) cells.
Methods K562 CML cells and bone marrow mononuclear cells(MNC)from patients with de novo CML and healthy donors were treated by various concentrations of TCA. The proliferation of K562 cells was detected by CCK-8 assay, Flow cytometry was employed to measure the cell cycle disribution of K562 cells, the apoptosis of K562 cells and primary CML cells, the mitochondrial transmembrane potential (△ψm) and Fas expression of K562 cells. Western blot assay was employed to quantify the expression of c-myc gene and the phosphrylation of Crkl in K562 cells treated by TCA. Real time PCR was employed to quantify the Bcr-Abl mRNA in K562 cells treated by TCA. Furthermore, the colony formation capability of CML BMMNC treated by TCA were determined by cytokines-riched methylcellulose culture assay.
Results TCA affected the proliferation of K562 cells in a time- and dose-dependent fashion. Low concentration of TCA (30μM) promoted the growth of K562 cells at 24 h and 48 h, while inhibited proliferation of K562 cells at 72 h, different from untreated control (P<0.01). Higher concentrations of TCA inhibited proliferation of K562 cells. The valueds of IC_(50) of TCA on K562 cells were 157.42μM at 24 h. Low concentration of TCA (30μM) could not induce apoptosis of K562 cells at any time. Middle concentration of TCA (60μM) induced just a small portion of cells undergoing apoptosis. High concentrations of TCA (≥90μM) leaded more than 10% HL60 cells to apoptosis. More apoptosis could been seen in K562 cells treated by increased concentrations of TCA and prolonged treating time. K562 cells were arrested at G2/M phase significantly by 60μM TCA while slightly by 30μM TCA at 24 h. c-Myc protein was highly expressed and Crkl were highly phosphorylated in K562 cells. TCA inhibited the expression of c-Myc and phosphorylation of Crkl in K562 cells in a time-and dose- dependent manner. TCA caused collapse of mitochondrial transmembrane potential and increased the expression of Fas in K562 cells in a time- and dose- dependent fashion. TCA inhibited the level Bcr-Abl mRNA in a dose dependent manner. TCA induced apoptosis of CML MNC dose dependently. Furthermore, TCA inhibited colony formation of CML MNC in a dose-depedent manner.
Conclusion Low concentration of TCA (30μM) exhibited pro-proliferation capability of K562 cells at 24 h and 48 h and anti-leukemia charateristic by inhibiting cell cycle progression. K562 cells treated by middle concentraion of TCA (60μM) were accumulated at G2 /M phase at 24 h and induced to apoptosis at 72 h. High concentraions of TCA (≥90μM ) inhibited proliferation of K562 cells by inducing K562 cells apoptosis in a time- and dose-dependent fashion. Two major death pathways, mitochondrial pathway and Fas pathway, were both involved in the apoptosis of K562 cells induced by TCA. TCA probabily induced apoptosis and proliferation inibition of CML cells by inhibiting the transcription of Bcr-Abl, which resulted in the decreased expression of BCR-ABL protein and diminished the effects of PTK, and subsequently downregulted expression of c-myc. In conclusion, TCA might be a promising drug with strong cytotoxicity on CML cells while little effects on normal BMMNC.
PARTⅢTrans-cinnamaldehyde induced differentiation of Myeloid Leukemia Cells
Objective To study the effects of trans-cinnamaldehyde (TCA) on the differentiation of myeloid leukemia cells.
Methods HL60 AML cells and K562 CML cells were treated by TCA and/or all-trans-Retinoic acid(ATRA). The morphology characteristic of cell treated by TCA was investigated by phase contrast microsope and stained by Wright’s-Gimsa. Flow cytometry was used to examine the differentiation antigens on TCA treated cells, the cell cycle distribution and apoptosis of TCA treated cells. Western blot assay was employed to quantify the expression of c-myc and p27 in HL60 cells and c-myc expression and phosphorylation level of Crkl in K562 cells. Confocal was employed to detect the expression of p16 and Cdc6 in HL60 cells.
Results TCA (≤60μM) induced HL60 to differentiate toward mature granule cells with the upregulation of CD11b on HL60 cells. TCA(≤60μM) induced K562 cells to differentiated toward monocytoid cells with CD14 and CD11b upregulated on K562 cells. Low concentration of TCA (20μM) arrested HL60 cells at G_0/G_1 phase at 72 h without significant apoptosis. Middle concentration of TCA(60μM) accumulated HL60 cells at G2/M at 24 h with the percentage of cells at G_0/G_1 phase decreasing. Middle concentration of TCA (60μM) induced significant apoptosis of HL60 cells at 48 h. Middle concentrations of TCA (60μM) increased the percentage of K562 cells at G2/M phase at 24 h and at 48 h, while decreased the percentage of G_2/M phase at 72 h with significant apoptosis. TCA induced HL60 cells differentiation accompanied by decreased expression of c-Myc protein and increased expression of p27 protein. The expression of c-Myc and phosphorylation level of Crkl in K562 cells decreased when K562 cells were induced to differentiation by TCA. TCA induced upregulated expression and translocation to nuclear of p16 but downregulated expression and translocation to cytoplasm of Cdc6 in differentiated HL60. TCA enhanced the differentiaiton of HL60 cells induction by ATRA.
Conclusion Low and middle concentrations of TCA induced myeloid cells to differentiate with different response in differente kind of cells and different concentraion of TCA. The differentiation of HL60 induced by TCA was related to depressed c-Myc protein and upregulated p27 protein. p16 and Cdc6 were involved in the differentiation of HL60 induced by TCA. The diferentiation effects induced by ATRA could be enhanced by TCA in HL60 cells. The differentiation of K562 cells induced by TCA was related to downregulation of c-Myc protein and phosphorylation level of Crkl protein.
PARTⅥEffects of Trans-cinnamaldehyde on the Adhesion and Migration of Acute Myeloid Leukemia Cells
Objective To study the effects of trans-cinnamaldehyde (TCA) on adhesion and migration of acute myeloid leukemia (AML) cells.
Methods Expression of CXCR4 on HL60 AML cells treated by TCA was detected by Flow cytometry. The effects of TCA on the migration and invasion of HL60 cells induced by recombinant human stromal cell derived factor-1α(rhSDF-1α) were investigated by transwell assay. HL60 cells were stained by carboxyfluorescein succinimidyl ester (CFSE). Staining of HL60 cells was determined by confocal and Flow cytometry. Confocal was also employed to detected the adhesion of CFSE stained HL60 cells co-cultured with AML bone marrow stromal cell (BMSC) treated by various concentraions of TCA. Secretion of SDF-1αwas quantified by ELISA. The morphologic characteristic of AML BMSC treated by TCA was oberved by phase contrast microscope. Flow cytometry was used to determine the apoptosis of AML BMSC.
Results Mean fluorescence intensity(MFI)of CXCR4 on HL60 cells treated by TCA decreased in a time- and dose-dependent manner. The migration and invasion of HL60 cells induced by rhSDF-1αwere inhibited significantly in dose dependently. Bright green fluorescence of CFSE was observed in the cytoplasm and nuclear of CFSE-stained HL60 cells, while no fluorescence could be observed in unstained HL60 cells and AML BMSC in the co-culture system. The adhesion of HL60 cells to the AML BMSC was inhibited by treatment of TCA for 6 h.in a dose-dependent manner. The secretion of SDF-1αby AML BMSC was depressed by TCA treatment for 24 h and 48 h. Furthermore, AML BMSC underwent apoptosis in a time- and dose-dependent manner after treated by TCA as evidenced by cell morphologic change and Flow cytometry.
Conclusion In addition to the direct effects on the leukmia cells, TCA impared the protection of AML BMSC and would contribute to the elimination of leukemia cells in bone marrow by inhibiting the chemoattraction and adhesion of leukemia cells to the BMSC via downregulating of CXCR4 expression on AML cells, inhibiting the secretion of SDF-1αby AML BMSC and inducing apoptosis of AML BMSC.
PARTⅤRegulation of mel18 gene in myeloid leukemia cells by Trans-cinnamaldehyde
Objective To investigate the expression and the significance of mel18 gene in acute myeloid leukemia (AML), the efffects of mel18 gene on the proliferation, apoptosis and cell cycle on the leukemia cells as well as the regulation of trans-cinnamaldehyde (TCA) on mel18 gene in AML cells.
Methods The expression of mel18 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR) in leukemia cell lines. Avidin-biotin-peroxidase complex (ABC) immunohistochemistry stain (IHC) was employed to detect Me118 protein in leukemia cell lines, bone marrow mononuclear cells(BMMNC)from patients with de novo AML and healthy donors as well as peripheral blood mononuclear cells (PBMNC) from healthy donors. The change of Mel18 expression was determined by Flow cytometry (FCM) and IHC in HL60 induced to differentiated by phorbol-12-myristate -13- acetaet (PMA). The plasmid pLenti6/V5-mel18 was constructed by gene-recombination technology and confirmed by sequencing. pLenti6/V5-mel18 and pLenti6/V5-LacZ (control) were transfected into the U937 and HL60 cells with Lipofectamine2000? with parent cells as the blank. The change of mel18 mRNA level in different groups was evaluated by RT- PCR while the Mel18 protein was determined by FCM. Proliferation of the cells was analyzed by CCK-8. Flow cytometry was employed to detect the apoptosis and cell cycle distribution. Mel18 expression in HL60 induced to differentiated by TCA was investigated by flow cytometry and immunohistochemistry.
Results K562 cells expresses mel18 mRNA strongly, HL60 cells expressed weakly and no mel18 mRNA was detected in U937 cells. Mel18 protein was detected in cytoplasm strong in K562 cells, weak in HL60 cells and normal BMMC. U937 cells doesnot express Mel18 protein. Expression of Mel18 protein couldnot be detected in 58.8 % of AML samples while could be detected in 100% of the normal BMMC samples. The expression of Mel18 increased in HL60 cells induced to differentiate by PMA. The sequence of the recombinant pLenti6/V5-mel18 was proved to be correct according to PCR and the sequence analysis. Expression of mel18 gene increased in U937 cells and HL60 cells after pLenti6/V5-mel18 transfection for 24 h. Transfection of pLenti6/V5-mel18 resulted in halted cell proliferation, increased apoptosis and increased the percentage of cells at G_0/G_1 phase in U937 cells and HL60 cells. The expression of Mel18 increased in HL60 cells and K562 cells undergoing differentiation induced by TCA without changes of location.
Conclusion The expression of mel18 gene decreased in AML cells and is correlated to cell differentiation. Overexpression of mel18 gene suppressed the proliferation, arrested cell cycle at G_0/G_1 phase and induced the apoptosis in leukemia cells, which meant mel18 could act as a antitumor gene in AML. TCA increased the expression of mel18 gene by inducing differentiation of leukemia cells.
目的:研究反式桂皮醛(trans-cinnamaldehyde,TCA)对急性髓细胞白血病(acute myeloid leukemia,AML)细胞增殖和凋亡的影响。
方法:用不同浓度的TCA和/或阿糖胞苷(β-D-Arabinofuranosyl cytosine,AraC)处理AML细胞株HL60或AML初治患者及健康人骨髓单个核细胞( bone marrow mononuclear cell,BMMNC)。CCK-8法测定HL60细胞增殖活性,流式细胞术检测HL60细胞周期变化, Annexin V/PI双标法检测HL60细胞和BMMNC凋亡,CD45/CD34/Annexin V三标法检测BMMNC CD34+细胞凋亡。Western blot检测TCA处理后HL60细胞c-myc基因表达。激光共聚焦术检测TCA处理后HL60细胞NF-κB活性。甲基纤维素法检测TCA处理后BMMNC克隆形成。
结果:TCA呈时间和剂量依赖性影响HL60细胞增殖。低浓度(10μM,20μM)TCA作用24 h后,促进HL60细胞增殖,和阴性对照组相比,有显著差异(P<0.05)。而较高浓度TCA对HL60细胞的增殖表现出抑制效应,并随浓度增加抑制作用明显增强。各浓度TCA作用48 h和72 h均表现出对HL60细胞增殖的抑制效应。TCA作用HL60细胞24 h,48 h和72 h的IC_(50)分别为78.40μM,58.13μM和50.31μM。高浓度TCA(≥80μM)可诱导>10%细胞发生凋亡,增加TCA的作用浓度使HL60细胞凋亡率进一步增加。TCA浓度≤60μM时作用24 h后,HL60细胞凋亡率<5%。在中浓度(40μM,60μM)TCA作用下,随作用时间延长, HL60细胞凋亡率也逐渐增加。低浓度(10μM,20μM)TCA始终不能显著增加凋亡率。中浓度TCA作用24 h后,HL60细胞明显阻滞于G2 /M。低浓度TCA作用24 h对细胞周期无明显影响,作用72 h后,细胞周期阻滞于G0/G1期。HL60细胞高表达c-Myc蛋白,TCA呈时间和剂量依赖性显著抑制c-Myc蛋白的表达。100μM TCA作用4 h后,HL60细胞NF-κB失活。TCA呈剂量依赖性诱导AML BMMNC和CD34+细胞凋亡,TCA对正常BMMNC和CD34+细胞的细胞毒作用很小,差异有显著性(P<0.05)。TCA非谱系特异性抑制AML原代细胞克隆形成。TCA协同AraC对HL60细胞、AML原代细胞及AML CD34+细胞的细胞毒性作用。
结论: TCA通过使NF-κB失活,下调原癌基因c-myc的表达,使HL60细胞周期阻滞和诱导细胞凋亡,发挥抗白血病效应。TCA对AML BMMNC和CD34+细胞也产生明显的凋亡诱导效应,并能协同AraC发挥抗白血病效应,对正常BMMNC的细胞毒作用很小。
第二部分反式桂皮醛对慢性髓细胞白血病细胞增殖和凋亡的影响
目的:研究反式桂皮醛(trans-cinnamaldehyde,TCA)对慢性髓细胞白血病(chronic myeloid leukemia,CML)细胞增殖和凋亡的影响。
方法:用不同浓度的TCA处理CML细胞株K562和初治CML患者及健康人骨髓单个核细胞(bone marrow mononuclear cell,BMMNC)。CCK-8法测定K562细胞增殖活性,流式细胞术检测K562细胞周期变化、K562细胞和BMMNC凋亡及K562细胞膜电位和Fas抗原表达。Western blot检测K562细胞c-myc基因表达和Crkl蛋白磷酸化水平。Real time PCR检测K562细胞Bcr-Abl mRNA表达。甲基纤维素法检测CML BMMNC克隆形成。
结果:TCA对K562的增殖作用的影响呈时间和剂量依赖性。低浓度(30μM)TCA作用24 h和48 h促进K562细胞增殖,和阴性对照组相比,有显著差异(P<0.01);作用72 h,抑制K562细胞增殖(P<0.01)。较高浓度TCA对K562细胞的增殖始终表现出抑制效应,并随浓度增加抑制作用明显增强。TCA处理K562细胞24 h的IC50是157.42μM。低浓度(30μM) TCA始终不能诱导K562细胞凋亡,中浓度(60μM)TCA作用24 h,仅极少部分细胞产生凋亡,与对照组细胞相比,差异无统计学意义(P>0.05)。高浓度TCA(≥90μM)作用24 h后可诱导>10%细胞发生凋亡,增加TCA浓度和作用时间可诱导更多细胞凋亡。60μM TCA作用24 h使K562细胞明显阻滞于G2 / M期,而30μM TCA则轻微增加G2 /M期细胞的比例(P>0.05)。K562细胞高表达c-Myc蛋白和磷酸化Crkl,TCA呈时间和剂量依赖性明显抑制c-Myc蛋白的表达和Crkl磷酸化水平。TCA呈时间和剂量依赖性下调线粒体跨膜电位和上调K562细胞的Fas表达。TCA呈剂量依赖性抑制K562细胞的Bcr-Abl转录水平。TCA呈剂量依赖性诱导CML BMMNC凋亡,对正常BMMNC的细胞毒作用很小。TCA非谱系特异性抑制CML BMMNC克隆形成。
结论:低浓度TCA(≤30μM)早期可能促进K562细胞增殖,而后期可诱导细胞周期阻滞,从而抑制K562细胞生长。中浓度TCA(60μM)早期阻滞细胞周期于G2 /M期,而随作用时间延长,诱导细胞出现凋亡。高浓度TCA(≥90μM)则通过诱导细胞凋亡,从而抑制K562细胞的生长,凋亡率呈时间和剂量依赖性。两条主要的凋亡通路,即线粒体介导和Fas介导的凋亡通路,都参与了TCA诱导的凋亡。TCA可能通过抑制Bcr-Abl转录,从而减少Bcr-Abl蛋白产物,削弱酪氨酸激酶效应,下调c-myc癌基因的蛋白表达,导致CML细胞凋亡、抑制CML细胞增殖。以上提示TCA具有明显的抗慢性髓细胞白血病效应,对正常骨髓单个核细胞的细胞毒作用很小,是一个很有应用前景的药。
第三部分反式桂皮醛诱导髓细胞白血病细胞分化
目的:本部分旨在研究反式桂皮醛(trans-cinnamaldehyde,TCA)对髓细胞白血病细胞分化的影响及机制。
方法:用TCA和/或反式维甲酸(all-trans-Retinoic acid,ATRA)处理髓细胞白血病细胞株HL60细胞及K562细胞。相差显微镜和Wright’s-Gimsa染色观察细胞形态变化。流式细胞术检测细胞分化抗原表达、细胞周期和凋亡。Western blot检测HL60细胞c-myc基因和p27基因表达、K562细胞c-myc基因表达和Crkl蛋白磷酸化水平。激光共聚焦术检测HL60细胞p16基因和Cdc6基因表达变化。
结果: TCA(≤60μM)诱导HL60细胞向成熟粒细胞分化,细胞表面分化抗原CD11b表达增加。TCA(≤60μM)诱导K562细胞向单核-巨噬细胞分化,细胞表面分化抗原CD14和CD11b表达均增加。低浓度(20μM)TCA作用72 h使HL60细胞阻滞于G0/G1期,不诱导明显细胞凋亡;中浓度(60μM)TCA作用24 h,HL60细胞G2/M期比例明显增加,G0/ G1期细胞比例明显下降,持续作用48 h时,HL60出现G2/M细胞比例降低,出现明显凋亡。中浓度(60μM)TCA作用于K562细胞24 h后,G2/M期细胞比例明显增加,持续作用48 h后,G2/M期细胞比例进一步增加,作用72 h时,K562细胞G2/M期比例下降,出现明显凋亡。TCA诱导HL60细胞分化伴原癌基因c-myc表达下降,细胞周期相关蛋白p27表达增强。TCA诱导K562细胞分化伴c-myc基因表达下调,Crkl蛋白磷酸化水平下降。TCA诱导HL60细胞分化还伴p16蛋白表达增加,向核内移位,而Cdc6蛋白表达下降,向核外迁移。TCA可协同增强ARTA对HL60细胞的诱导分化效应。
结论: TCA(≤60μM)可诱导髓细胞白血病细胞分化,但不同种类的细胞对TCA的反应有差异性,而且同种白血病细胞对不同浓度的TCA的反应不同。TCA诱导HL60白血病细胞分化与c-myc蛋白水平下降和p27蛋白水平上调有关,p16基因和Cdc6基因参与TCA诱导的HL60细胞分化过程。TCA可增强ATRA对HL60细胞的诱导分化效应。TCA诱导K562细胞的分化与c-myc和Crkl蛋白磷酸化水平下降有关。
第四部分反式桂皮醛对急性髓细胞白血病细胞粘附和迁移的影响
目的:本部分旨在研究反式桂皮醛(trans-cinnamaldehyde,TCA)对急性髓细胞白血病(acute myeloid leukemia,AML)细胞粘附和迁移的影响。
方法:TCA处理AML细胞株HL60细胞,流式检测细胞表面CXCR4的表达。Transwell法检测不同浓度TCA对重组基质细胞衍生因子-1α(stromal cell derived factor-1α,SDF-1α)诱导的HL60迁移和浸润的影响。羧基荧光素二乙酸盐琥珀酰亚胺酯(carboxyfluorescein succinimidyl ester,CFSE)标记HL60细胞,共聚焦显微镜和流式细胞术检测HL60细胞染色情况。CFSE标记的HL60细胞与AML骨髓基质细胞(bone marrow stromal cell, BMSC)共培养,用共聚焦检测不同浓度TCA处理后HL60细胞与AML BMSC的粘附情况。ELISA法检测TCA对AML BMSC分泌SDF-1α的影响。相差显微镜观察TCA处理后AML BMSC细胞形态变化,流式细胞术检测AML BMSC细胞凋亡。
结果:TCA作用后,HL细胞CXCR4表达的平均荧光强度(mean fluorescence intensity,MFI)呈时间和剂量依赖性明显下降(P值均<0.05)。TCA呈剂量依赖性抑制HL60趋向重组人SDF-1α的迁移和浸润。CFSE标记后培养过夜的HL60细胞发出很强的绿色强荧光,在细胞核和细胞质内均有分布,而未标记的HL60细胞和BMSC未见荧光。TCA作用6 h后呈剂量依赖性抑制HL60细胞与AML BMSC间的粘附。TCA呈剂量依赖性抑制AML BMSC分泌SDF-1α。TCA作用24 h,AML BMSC细胞间隙增大。TCA呈现时间和剂量依赖性诱导AML BMSC凋亡。
结论:TCA除可直接抑制白血病细胞的增殖、诱导白血病细胞凋亡和分化外,还可能通过下调白血病细胞CXCR4表达、抑制骨髓基质细胞SDF-1分泌和诱导骨髓基质细胞凋亡、抑制白血病细胞对骨髓基质细胞的趋化,使白血病细胞一方面失去基质层的支持,另一方面易于发生凋亡,因此有可能削弱骨髓基质细胞对白血病细胞潜在的保护作用,有利于清除髓内白血病细胞。
第五部分反式桂皮醛调节髓细胞白血病细胞mel18基因表达
目的:本研究旨在检测mel18基因在急性髓细胞白血病(acute myeloid leukemia,AML)的表达,研究mel18基因对AML细胞的增殖、凋亡和周期的影响,探讨反式桂皮醛(trans-cinnamaldehyde,TCA)对AML细胞mel18基因的调控。
方法:运用逆转录聚合酶链式反应(reverse transcription polymerase chain reaction, RT-PCR)检测白血病细胞株mel18 mRNA表达,生物素-生物素-过氧化物酶(avidin-biotin-peroxidase complex,ABC)法免疫组织化学(immunohistochemistry stain,IHC)染色分析髓细胞白血病细胞株、初治AML患者骨髓单个核细胞和健康人外周血及骨髓单个核细胞的Mel18蛋白表达。运用十四酰佛波乙酸酯(phorbol-12-myristate-13-acetaet,PMA)诱导HL60细胞分化,流式细胞术、免疫组化法和激光共聚焦术检测诱导分化细胞的Mel18蛋白表达变化。通过基因重组和分子克隆技术构建pLenti6/V5-mel18真核表达载体并鉴定,应用脂质体(Lipofectamine2000?)转染,将pLenti6/V5-mel18和pLenti6/V5-LacZ(阴性对照)导入HL60和U937细胞。RT- PCR和流式细胞术检测pLenti6/V5-mel18质粒转染细胞中mel8基因的表达。用CCK-8法测定细胞增殖活性,流式细胞术检测细胞凋亡和细胞周期。TCA诱导K562细胞和HL60细胞分化,流式细胞术和激光共聚焦术检测Mel18蛋白表达。
结果:mel18 mRNA在K562细胞强表达,HL60细胞弱表达,U937细胞无表达。Mel18蛋白主要表达在细胞质中,在K562细胞和正常外周血中高表达,HL60和正常骨髓单个核细胞中低表达,而U937细胞不表达Mel18蛋白。Mel18蛋白在AML骨髓中缺失率为58.8 %,正常对照骨髓单个核细胞中未检测到Mel18蛋白缺失。HL60细胞被PMA诱导分化后Mel18蛋白表达增加,亚细胞定位不变。重组质粒pLenti6/V5-mel18经过PCR检测和公司测序证实构建成功。pLenti6/V5-mel18质粒转染24 h后, U937细胞和HL60细胞的mel18基因表达增加,转染pLenti6/V5-mel18的U937细胞和HL60细胞增殖受抑,细胞出现凋亡,G0/G1期细胞比例增加。TCA诱导HL60细胞和K562细胞分化伴Mel18蛋白表达增加,Mel18亚细胞定位不发生改变。
结论: mel18基因在AML中表达降低,mel18基因的表达与分化相关。mel18在AML中可能发挥抑癌基因的作用,增强mel18基因的表达可抑制AML细胞株增殖,诱导细胞凋亡,使细胞周期阻滞。TCA诱导白血病细胞分化可增加mel18基因的表达。
PART I Effects of Trans-cinnamaldehyde on the Proliferation and Apoptosis of Acute Myeloid Leukemia Cells
Objective To study the effects of trans-cinnamaldehyde (TCA) on the proliferation and apoptosis of acute myeloid leukemia (AML) cells.
Methods HL60 AML cells and bone marrow mononuclear cells(BMMNC)from patients with de novo AML and healthy donors were treated by various concentrations of TCA and / orβ-D-Arabinofuranosyl cytosine (AraC). The proliferation of HL60 cells were detected by CCK-8 assay. Flow cytometry was empolyed to detect the cell cycle disribution of HL60 cells. The apoptosis of HL60 cells and BMMNC was measured by Annexin V/PI double labeled, and the apoptosis of BMMNC CD34+ cells were examined by triple labled with CD45/CD34/Annexin V. Western blot assay was employed to quantify the expression of c-myc gene in HL60 cells treated by TCA.The activity of NF-κB was investigated by confocal. Furthermore, the colony formation capability of BMMNC treated by TCA were determined by cytokines-riched methylcellulose culture assay.
Results TCA affected the proliferation of HL60 cells in a time- and dose-dependent fashion. Low concentrations of TCA (10μM, 20μM) promoted the growth of HL60 cells at 24 h, different from untreated control significantly (P<0.05). Higher concentrations of TCA inhibited proliferation of HL60 cells. TCA administrated at any concentraion halted proliferation of HL60 cells at 48 h and 72 h. The values of IC_(50) of TCA on HL60 cells were 78.40μM, 58.13μM and 50.31μM at 24 h, 48 h and 72 h, respectively. High concentrations of TCA (≥80μM) induced more than 10% cells to undergo apoptosis. More apoptosis could be seen in HL60 cells treated by increased concentrations of TCA. Less than 5% HL60 cells underwent apoptosis when treated by TCA with the concentration no more than 60μM for 24 h. The apoptosis of HL60 cells treated by middle concentrations of TCA (40μM, 60μM) increased when the treated time was prolonged. Low concentrations of TCA (10μM, 20μM) could not induce significant apoptosis of HL60 cells at any time. HL60 cells were arrested at G2 /M phase after treated by middle concentraions of TCA for 24 h. Low concentraions of TCA did not affect the cell cycle of HL60 cells at 24 h but accumulated HL60 cell at G2 /M phase at 72 h. c-Myc protein was highly expressed in HL60 cells and TCA inhibited the expression of c-Myc in HL60 cells in a time- and dose- dependent manner. The activity of NF-κB in HL60 cells was inhibited by treatment of 100μM TCA for 4 h. TCA induced apoptosis of AML BMMNC and AML CD34+ cells time- and dose-dependently.The cytotoxicity of TCA on normal BMMNC was slight. TCA inhibited colony formation of AML BMMNC. Furthermore, TCA synergized the cytotoxicity of AraC on HL60 cells, AML BMMNC and AML BMMNC CD34+ cells as well.
Conclusion TCA exhibited anti-leukemia charateristic by inhibiting cell cycle progression, inducing apoptosis, deactivating NF-κB and repressing the expression of c-myc gene. TCA leaded AML BMMNC cells and AML CD34+ cells to significantly apoptosis with little cytotoxicity on normal BMMNC. Moreover, TCA synergized the anti-leukemia effect of AraC.
PARTⅡEffects of Trans-cinnamaldehyde on the Proliferation and Apoptosis of Chronic Myeloid Leukemia Cells
Objective To study the effects of trans-cinnamaldehyde (TCA) on the proliferation and apoptosis of chronic myeloid leukemia (CML) cells.
Methods K562 CML cells and bone marrow mononuclear cells(MNC)from patients with de novo CML and healthy donors were treated by various concentrations of TCA. The proliferation of K562 cells was detected by CCK-8 assay, Flow cytometry was employed to measure the cell cycle disribution of K562 cells, the apoptosis of K562 cells and primary CML cells, the mitochondrial transmembrane potential (△ψm) and Fas expression of K562 cells. Western blot assay was employed to quantify the expression of c-myc gene and the phosphrylation of Crkl in K562 cells treated by TCA. Real time PCR was employed to quantify the Bcr-Abl mRNA in K562 cells treated by TCA. Furthermore, the colony formation capability of CML BMMNC treated by TCA were determined by cytokines-riched methylcellulose culture assay.
Results TCA affected the proliferation of K562 cells in a time- and dose-dependent fashion. Low concentration of TCA (30μM) promoted the growth of K562 cells at 24 h and 48 h, while inhibited proliferation of K562 cells at 72 h, different from untreated control (P<0.01). Higher concentrations of TCA inhibited proliferation of K562 cells. The valueds of IC_(50) of TCA on K562 cells were 157.42μM at 24 h. Low concentration of TCA (30μM) could not induce apoptosis of K562 cells at any time. Middle concentration of TCA (60μM) induced just a small portion of cells undergoing apoptosis. High concentrations of TCA (≥90μM) leaded more than 10% HL60 cells to apoptosis. More apoptosis could been seen in K562 cells treated by increased concentrations of TCA and prolonged treating time. K562 cells were arrested at G2/M phase significantly by 60μM TCA while slightly by 30μM TCA at 24 h. c-Myc protein was highly expressed and Crkl were highly phosphorylated in K562 cells. TCA inhibited the expression of c-Myc and phosphorylation of Crkl in K562 cells in a time-and dose- dependent manner. TCA caused collapse of mitochondrial transmembrane potential and increased the expression of Fas in K562 cells in a time- and dose- dependent fashion. TCA inhibited the level Bcr-Abl mRNA in a dose dependent manner. TCA induced apoptosis of CML MNC dose dependently. Furthermore, TCA inhibited colony formation of CML MNC in a dose-depedent manner.
Conclusion Low concentration of TCA (30μM) exhibited pro-proliferation capability of K562 cells at 24 h and 48 h and anti-leukemia charateristic by inhibiting cell cycle progression. K562 cells treated by middle concentraion of TCA (60μM) were accumulated at G2 /M phase at 24 h and induced to apoptosis at 72 h. High concentraions of TCA (≥90μM ) inhibited proliferation of K562 cells by inducing K562 cells apoptosis in a time- and dose-dependent fashion. Two major death pathways, mitochondrial pathway and Fas pathway, were both involved in the apoptosis of K562 cells induced by TCA. TCA probabily induced apoptosis and proliferation inibition of CML cells by inhibiting the transcription of Bcr-Abl, which resulted in the decreased expression of BCR-ABL protein and diminished the effects of PTK, and subsequently downregulted expression of c-myc. In conclusion, TCA might be a promising drug with strong cytotoxicity on CML cells while little effects on normal BMMNC.
PARTⅢTrans-cinnamaldehyde induced differentiation of Myeloid Leukemia Cells
Objective To study the effects of trans-cinnamaldehyde (TCA) on the differentiation of myeloid leukemia cells.
Methods HL60 AML cells and K562 CML cells were treated by TCA and/or all-trans-Retinoic acid(ATRA). The morphology characteristic of cell treated by TCA was investigated by phase contrast microsope and stained by Wright’s-Gimsa. Flow cytometry was used to examine the differentiation antigens on TCA treated cells, the cell cycle distribution and apoptosis of TCA treated cells. Western blot assay was employed to quantify the expression of c-myc and p27 in HL60 cells and c-myc expression and phosphorylation level of Crkl in K562 cells. Confocal was employed to detect the expression of p16 and Cdc6 in HL60 cells.
Results TCA (≤60μM) induced HL60 to differentiate toward mature granule cells with the upregulation of CD11b on HL60 cells. TCA(≤60μM) induced K562 cells to differentiated toward monocytoid cells with CD14 and CD11b upregulated on K562 cells. Low concentration of TCA (20μM) arrested HL60 cells at G_0/G_1 phase at 72 h without significant apoptosis. Middle concentration of TCA(60μM) accumulated HL60 cells at G2/M at 24 h with the percentage of cells at G_0/G_1 phase decreasing. Middle concentration of TCA (60μM) induced significant apoptosis of HL60 cells at 48 h. Middle concentrations of TCA (60μM) increased the percentage of K562 cells at G2/M phase at 24 h and at 48 h, while decreased the percentage of G_2/M phase at 72 h with significant apoptosis. TCA induced HL60 cells differentiation accompanied by decreased expression of c-Myc protein and increased expression of p27 protein. The expression of c-Myc and phosphorylation level of Crkl in K562 cells decreased when K562 cells were induced to differentiation by TCA. TCA induced upregulated expression and translocation to nuclear of p16 but downregulated expression and translocation to cytoplasm of Cdc6 in differentiated HL60. TCA enhanced the differentiaiton of HL60 cells induction by ATRA.
Conclusion Low and middle concentrations of TCA induced myeloid cells to differentiate with different response in differente kind of cells and different concentraion of TCA. The differentiation of HL60 induced by TCA was related to depressed c-Myc protein and upregulated p27 protein. p16 and Cdc6 were involved in the differentiation of HL60 induced by TCA. The diferentiation effects induced by ATRA could be enhanced by TCA in HL60 cells. The differentiation of K562 cells induced by TCA was related to downregulation of c-Myc protein and phosphorylation level of Crkl protein.
PARTⅥEffects of Trans-cinnamaldehyde on the Adhesion and Migration of Acute Myeloid Leukemia Cells
Objective To study the effects of trans-cinnamaldehyde (TCA) on adhesion and migration of acute myeloid leukemia (AML) cells.
Methods Expression of CXCR4 on HL60 AML cells treated by TCA was detected by Flow cytometry. The effects of TCA on the migration and invasion of HL60 cells induced by recombinant human stromal cell derived factor-1α(rhSDF-1α) were investigated by transwell assay. HL60 cells were stained by carboxyfluorescein succinimidyl ester (CFSE). Staining of HL60 cells was determined by confocal and Flow cytometry. Confocal was also employed to detected the adhesion of CFSE stained HL60 cells co-cultured with AML bone marrow stromal cell (BMSC) treated by various concentraions of TCA. Secretion of SDF-1αwas quantified by ELISA. The morphologic characteristic of AML BMSC treated by TCA was oberved by phase contrast microscope. Flow cytometry was used to determine the apoptosis of AML BMSC.
Results Mean fluorescence intensity(MFI)of CXCR4 on HL60 cells treated by TCA decreased in a time- and dose-dependent manner. The migration and invasion of HL60 cells induced by rhSDF-1αwere inhibited significantly in dose dependently. Bright green fluorescence of CFSE was observed in the cytoplasm and nuclear of CFSE-stained HL60 cells, while no fluorescence could be observed in unstained HL60 cells and AML BMSC in the co-culture system. The adhesion of HL60 cells to the AML BMSC was inhibited by treatment of TCA for 6 h.in a dose-dependent manner. The secretion of SDF-1αby AML BMSC was depressed by TCA treatment for 24 h and 48 h. Furthermore, AML BMSC underwent apoptosis in a time- and dose-dependent manner after treated by TCA as evidenced by cell morphologic change and Flow cytometry.
Conclusion In addition to the direct effects on the leukmia cells, TCA impared the protection of AML BMSC and would contribute to the elimination of leukemia cells in bone marrow by inhibiting the chemoattraction and adhesion of leukemia cells to the BMSC via downregulating of CXCR4 expression on AML cells, inhibiting the secretion of SDF-1αby AML BMSC and inducing apoptosis of AML BMSC.
PARTⅤRegulation of mel18 gene in myeloid leukemia cells by Trans-cinnamaldehyde
Objective To investigate the expression and the significance of mel18 gene in acute myeloid leukemia (AML), the efffects of mel18 gene on the proliferation, apoptosis and cell cycle on the leukemia cells as well as the regulation of trans-cinnamaldehyde (TCA) on mel18 gene in AML cells.
Methods The expression of mel18 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR) in leukemia cell lines. Avidin-biotin-peroxidase complex (ABC) immunohistochemistry stain (IHC) was employed to detect Me118 protein in leukemia cell lines, bone marrow mononuclear cells(BMMNC)from patients with de novo AML and healthy donors as well as peripheral blood mononuclear cells (PBMNC) from healthy donors. The change of Mel18 expression was determined by Flow cytometry (FCM) and IHC in HL60 induced to differentiated by phorbol-12-myristate -13- acetaet (PMA). The plasmid pLenti6/V5-mel18 was constructed by gene-recombination technology and confirmed by sequencing. pLenti6/V5-mel18 and pLenti6/V5-LacZ (control) were transfected into the U937 and HL60 cells with Lipofectamine2000? with parent cells as the blank. The change of mel18 mRNA level in different groups was evaluated by RT- PCR while the Mel18 protein was determined by FCM. Proliferation of the cells was analyzed by CCK-8. Flow cytometry was employed to detect the apoptosis and cell cycle distribution. Mel18 expression in HL60 induced to differentiated by TCA was investigated by flow cytometry and immunohistochemistry.
Results K562 cells expresses mel18 mRNA strongly, HL60 cells expressed weakly and no mel18 mRNA was detected in U937 cells. Mel18 protein was detected in cytoplasm strong in K562 cells, weak in HL60 cells and normal BMMC. U937 cells doesnot express Mel18 protein. Expression of Mel18 protein couldnot be detected in 58.8 % of AML samples while could be detected in 100% of the normal BMMC samples. The expression of Mel18 increased in HL60 cells induced to differentiate by PMA. The sequence of the recombinant pLenti6/V5-mel18 was proved to be correct according to PCR and the sequence analysis. Expression of mel18 gene increased in U937 cells and HL60 cells after pLenti6/V5-mel18 transfection for 24 h. Transfection of pLenti6/V5-mel18 resulted in halted cell proliferation, increased apoptosis and increased the percentage of cells at G_0/G_1 phase in U937 cells and HL60 cells. The expression of Mel18 increased in HL60 cells and K562 cells undergoing differentiation induced by TCA without changes of location.
Conclusion The expression of mel18 gene decreased in AML cells and is correlated to cell differentiation. Overexpression of mel18 gene suppressed the proliferation, arrested cell cycle at G_0/G_1 phase and induced the apoptosis in leukemia cells, which meant mel18 could act as a antitumor gene in AML. TCA increased the expression of mel18 gene by inducing differentiation of leukemia cells.
引文
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