【作者】 周文静；

【导师】 金洁；

【作者基本信息】 浙江大学 ， 内科血液学， 2008， 博士

【摘要】 急性髓系白血病（acute myeloid leukemia,AML）是一组异源性的造血祖细胞克隆性疾病,肿瘤细胞具有髓系细胞的特征,但失去正常分化能力并异常增殖。骨髓增生异常综合征（myelodysplastic syndromes,MDS）是一组起源于造血系统定向干细胞或多能干细胞的异质性克隆性疾病,以外周血白细胞减少,骨髓出现病态造血为特点,并可转化为急性白血病。虽然目前联合化疗、造血干细胞移植及新的针对分子靶点的治疗取得一定的成效,但仍迫切需要研究新的AML和MDS有效治疗药物。柳氮磺胺吡啶（sufasalazine,SSZ）是一种合成的抗炎药物,常规用于治疗炎症性肠病和风湿性关节炎。近期研究证实:SSZ具有抗肿瘤作用,可抑制多种人肿瘤细胞株生长并诱导凋亡。同时,关于脑胶质瘤的体内研究也证实SSZ具有抗肿瘤作用并已开展1-2期临床试验。但其对AML及MDS的作用还未见报道。本实验旨在研究SSZ对人AML细胞和MDS细胞株MUTZ-1的作用并初步探讨其作用机制,以期开发新的AML和MDS有效治疗药物。首先,我们采用MTT法研究SSZ对人AML细胞的生长抑制作用。结果显示:不同浓度SSZ作用人AML细胞株KASUMI-1、NB-4、K-562、U-937、KG-1细胞一定时间后,可抑制细胞生长并呈浓度依赖性,48h的半数抑制浓度IC₅₀值分别为:0.372mM、0.302mM、0.734mM、1.043mM、0.824mM。同时,SSZ也能抑制8例原代AML细胞的生长并呈浓度依赖性,48h的IC₅₀值为0.4—1.2mM。SSZ对正常人骨髓单个核细胞的生长抑制作用相对较弱,48h的IC₅₀值大于2.4mM。同时,我们进一步探讨SSZ作用AML细胞的机制。采用瑞氏——姬姆萨染色并在油镜下观察SSZ作用前后细胞形态学变化,结果显示:SSZ 1.5mM作用AML细胞株KASUMI-1、NB-4 48h后细胞出现典型的凋亡形态学特征:如胞浆空泡化、细胞体积缩小、细胞核染色质凝聚、边缘化、形成新月型核或环形核、细胞核碎裂、凋亡小体形成等。进一步采用AnnexinV/PI双染流式细胞仪检测细胞凋亡,结果显示:不同浓度SSZ作用KASUMI-1、NB-4细胞24h,可诱导细胞凋亡并呈浓度依赖性。我们进一步研究线粒体途径在SSZ诱导AML细胞凋亡中的作用。结果发现:不同浓度SSZ作用KASUMI-1、NB-4细胞24h,可诱导细胞线粒体膜电位下降并呈浓度依赖性。同时,Western Blot法检测不同浓度SSZ作用24h后AML细胞株KASUMI-1细胞中线粒体途径相关凋亡执行蛋白Caspase-9、Caspase-3、PARP的变化,结果显示:随着药物作用浓度的增加,出现Caspase-9、Caspase-3、PARP的活化。由此提示:线粒体途径在SSZ诱导的AML细胞株KASUMI-1细胞凋亡中起重要作用。此外,Western Blot法检测SSZ作用后AML细胞株KASUMI-1细胞中凋亡调节蛋白Bcl-2家族、IAPs家族的成员变化。结果显示:不同浓度SSZ作用KASUMI-1细胞24h,可改变Bcl-2/Bax及Bcl-xl/Bax的表达比例并与药物作用浓度呈负相关;同时,IAPs家族成员XIAP、cIAP-1、cLAP-2的表达随着药物作用浓度增加而下调,蛋白表达水平与药物作用浓度负相关。从而提示:凋亡调节蛋白Bcl-2家族、IAPs家族参与SSZ诱导的AML细胞凋亡。同时,Western Blot检测不同浓度SSZ作用KASUMI-1细胞24h后细胞核中NF-kB的表达,结果显示:细胞核NF-kB蛋白表达水平与药物作用浓度负相关,间接证实NF-kB抑制可能为SSZ发挥抗AML作用的又一重要机制。此外,我们的研究证实不同浓度SSZ作用KASUMI-1细胞24h可下调AML1-ETO融合蛋白的表达,蛋白表达水平与药物作用浓度负相关,提示AML1-ETO可能为SSZ发挥抗AML作用的重要靶点。其次,我们研究了SSZ对人MDS细胞株MUTZ-1的作用。结果显示:SSZ可有效抑制MUTZ-1细胞的生长并具有浓度依赖的特点,SSZ作用MUTZ-1 48h的IC₅₀值为0.672mM。此外,AnnexinV/PI双染流式细胞仪检测发现:不同浓度SSZ作用MUTZ-1 24h可诱导细胞的凋亡,并呈浓度依赖性。同时证实:SSZ能诱导MUTZ-1细胞线粒体膜电位下降,并可激活Caspase-3。提示SSZ诱导MUTZ-1凋亡的机制可能与线粒体膜电位下降和Caspase-3活化有关。综上所述,我们的研究证实:SSZ具有潜在的抗AML及抗MDS效应,可能成为治疗AML及MDS的新药。更多还原

【Abstract】 Acute myeloid leukemia（AML）is a heterogenous clonal disorder of haemopoietic progenitor cells,which lose the ability to differentiate normally and to respond to normal regulators of proliferation.Myelodysplastic syndromes（MDS）are a group of disorders characterized by progressive cytopenias and transformation to acute leukemia.Although the recent development of new treatment strategies have made progress, new strategies are still urgently needed both in AML and in MDS.Sulfasalazine（SSZ）is a synthetic anti-inflammatory drug and routinely used for the treatment of inflammatory bowel disease and rheumatoid arthritis.Recently,SSZ demonstrated anti-cancer properties on several human tumor cell lines and in vivo studies also confirmed its anti-cancer properites in glioblastomas.In the present study,we investigated the effects and mechanisms of SSZ in human AML cells and MDS cell line MUTZ-1.Firstly,we examined the effect of SSZ on human AML cells.Using MTT assay,we examined the effects of different concentrations of SSZ on the growth of human AML cells.The results indicated that cell viability in the presence of SSZ decreased in a dose-dependent manner.The inhibitory rates of cell growth were positively correlated with SSZ concentrations.The growth-inhibitory IC₅₀values were 0.372mM、0.302mM、0.734mM、1.043mM、0.824mM in KASUMI-1、NB-4、K-562、U-937、KG-1 AML cells after the treatment of SSZ for 48 hours,respectively.SSZ also inhibited the growth of freshly isolated AML cells from 8 patients with an IC₅₀（at 48h）of 0.4-1.2mM.In contrast,the IC₅₀of SSZ was more than 2.4mM in normal cells.To explore the possible mechanism of the anti-leukemic activity of SSZ in AML cells,further investigation was carried out.In order to investigate whether apoptosis is associated with the anti-leukemic activity of SSZ in AML cells,we observed the cell morphology using Wright-Giemsa staining,evaluated the exposure of phosphatidylserine（PS）on KASUMI-1 and NB-4 cells after double staining with fluorescein isothiocyanate（FITC）-labeled annexin V and propidium iodide（PI）.We found typical apoptotic morphology after KASUMI-1 and NB-4 cells were exposed to SSZ for 48hr.And Annexin V/PI staining also confirmed SSZ induced apoptosis in KASUMI-1 and NB-4 cells in a dose-dependent manner.In order to explore the possible mechanism and pathway of the signal conduction of the cell apoptosis induced by SSZ,we examined mitochondrial transmembrane potential and revealed that SSZ led to mitochondrial dysfunction in KASUMI-1 and NB-4 cells.Besides,western blot confirmed that Caspase-9,Caspase -3 and PARP were activated in KASUMI-1 cell following treatment with 0.5-1.5mM SSZ for 24h.These results indicate that mitochondrial pathway may play an important role in ssz induced apoptosis in KASUMI-1 cell.Meanwhile,the role of Bcl-2 family and IAPs family members were also investigated by western blot when 0.5-1.5mM SSZ was given for 24h in KASUMI-1 cell,the results showed that Bcl-2/Bax and Bcl-xl/Bax ratio were negatively correlated with SSZ concentrations,and the IAPs family members XIAP,cIAP-1,cIAP-2 protein levels were also negatively correlated with SSZ concentrations.These results indicate that both Bcl-2 family and IAPs family were involved in ssz induced apoptosis in KASUMI-1 cell.We also found an decrease of nuclear NF-kB（p65）expression in KASUMI-1 cell following treatment with 0.5-1.5mM SSZ for 24h by western blot which indirectly indicated that NF-kB inhibition may be an important mechanism for anti-leukemic activity of SSZ in KASUMI-1 cell. Meanwhile,we confirmed that AML1-ETO fusion protein which carried by KASUMI-1 cell was also down-regulated with 0.5-1.5mM SSZ for 24h by western blot which indicated that AML-ETO may be another important target for SSZ in KASUMI-1 cell.Secondly,we examined the effect of SSZ on human MDS cell line MUTZ-1.MTT assay revealed that MUTZ-1 cell viability in the presence of SSZ decreased in a dose-dependent manner.The inhibitory rates of cell growth were positively correlated with SSZ concentrations.The growth-inhibitory IC₅₀values was 0.672mM after the treatment of SSZ for 48h.Besides,Annexin V/PI staining confirmed SSZ induced apoptosis in MUTZ-1 cell in a dose dependent manner.Further more,JC-1 staining revealed mitochondrial dysfunction in MUTZ-1 cell after 1.5mM SSZ treatment for 24h.Western blot revealed activation of caspase-3 when 0.5-1.5mM SSZ was treated for 24h in MUTZ-1 cell.These results indicated that SSZ can inhibit the viability and induce apoptosis of MUTZ-1 cell,dissipation of mitochondrial transmembrane and activating of Caspase-3 were involved in this process.In conclusion,we found out that SSZ had potent anti-leukemic activity in AML and anti-MDS activity in MUTZ-1 cell in vitro,suggesting that further investigation of this agent is warranted.更多还原