CN105902545A - Soimycin A用于制备白血病放化疗增敏剂的用途 - Google Patents
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Abstract
本发明涉及一种Soimycin A制备急性髓性白血病(Acute myeloid leukemia，AML)放化疗增敏剂的用途。本发明的体外及动物实验表明，Soimycin A具有提高AML细胞系HL‑60及慢性髓性白血病(Chromic myeloid leukemia，CML)细胞系K562放疗敏感性的作用，并证实Soimycin A具有提高HL‑60细胞移植构建的AML小鼠化疗后存活率的作用。本发明所述增敏剂用于急性髓性白血病放化疗的应用前景好。
Description
技术领域
本发明涉及Soimycin A用于制备急性髓性白血病放化疗增敏剂的用途
背景技术
急性髓性白血病(Acute myeloid leukemia，AML)是白血病中最常见的一类恶性克隆性疾病，AML主要表现为造血干细胞异常增殖、分化障碍、凋亡受阻而停滞于不完全成熟的各阶段，并广泛侵润骨髓及其它器官和组织。现阶段急性髓性白血病的治疗以化疗为主，辅以放疗。化疗药物主要通过干扰DNA合成，抑制肿瘤细胞快速增殖分裂；放射性治疗通过电离辐射产生大量能量，进而破坏肿瘤细胞染色体结构，使细胞生长停止。尽管近年来新的化疗药物不断出现，放化疗方案不断改进，但仍有50～80％的AML患者在获得完全缓解后出现复发现象，并最终发展为难治性白血病。究其原因主要在于肿瘤细胞产生耐药性及放射性抵抗。
放化疗增敏是指采用微小剂量的该物质与放疗/化疗联合使用即能显著提高放化疗效果，在肿瘤治疗中低毒高效的放化疗增敏剂具有广泛的应用前景。因此，深入探究AML肿瘤细胞耐药性及放射性抵抗的分子机制，并寻找高效放化疗增敏剂是治疗AML亟待解决的问题之一。
研究发现肿瘤细胞耐药性及放射性抵抗的产生与一系列基因被诱导表达紧密相关。FoxM1(Forkhead-box M1)是转录因子forkhead家族成员之一。FoxM1在调节细胞增殖，细胞周期进程，分化，DNA损伤修复及细胞凋亡中发挥重要作用。FoxM1是实体瘤中最常见表达上调的基因之一，在结肠癌、肝癌、前列腺癌和乳腺癌等多种肿瘤中检测到其高表达，说明FoxM1与肿瘤发生具有紧密联系。最新的研究发现，FoxM1介导了部分肿瘤细胞的耐药性，干扰FoxM1的表达后肿瘤细胞化疗敏感性增加。但FoxM1是否介导了AML肿瘤细胞耐药性及放射性抵抗仍不明晰，FoxM1是否可作为AML放化疗增敏剂的干预靶点尚不清楚。
Soimycin A是一种天然噻唑类抗生素，其分子式为C71H81N19O18S5，分子量为1648.84，并具有以下结构
研究证实Soimycin A可以抑制FoxM1在mRNA及蛋白水平的表达，并影响FoxM1下游靶基因的转录。Soimycin A对AML肿瘤细胞的影响尚不清楚，我们在探究FoxM1基因对小鼠骨髓细胞耐药性(5-氟尿嘧啶核苷)的影响研究中，发现Soimycin A能够提高AML细胞系放疗敏感性及AML小鼠化疗敏感性，该结果目前尚未见国内外报道。
发明内容
本发明的目的是提供一种针对FoxM1靶点的Soimycin A用于制备AML放化疗增敏剂的用途，该增敏剂提高AML细胞系放疗敏感性及AML小鼠化疗后生存率疗效明显。在目前临床上缺乏高效AML放化疗增敏剂的前提下，Soimycin A应用前景好。
本发明的技术方案是：
Soimycin A在制备急性髓性白血病放化疗增敏剂中的应用。
所述白血病是急性髓性白血病。
所述增敏剂通过抑制FoxM1的表达提高AML细胞系放疗及AML小鼠化疗敏感性。
本发明所述增敏剂，可不经过口服给药，以注射剂或输液剂等形式给药，给药量以每公斤体重每12小时20～40mg为宜。
发明人经研究发现所述Soimycin A在体外对AML细胞系HL-60及CML细胞系K562具有提高放疗敏感性的作用。结合小鼠实验，在AML小鼠模型中观察证实Soimycin A具有提高化疗敏感性的作用。
Soimycin A作为一种已知化合物可通过本领域已知方法制备或从市场上获得，因此来源方便，成本较低。
以下的实验实施例是对本发明的进一步详细说明但不意味着对本发明的限制。
附图说明
图1为5-氟尿嘧啶对FoxM1基因敲除型小鼠生存率的影响；
图2为Soimycin A预处理对髓性白血病细胞系放疗后凋亡率的影响；
图3为Soimycin A预处理对AML小鼠化疗后生存率的影响。
具体实施方式
人AML细胞系HL-60及CML细胞系K562购自ATCC公司；
NSGS受体小鼠购自中国人民解放军第三军医大学实验动物中心；
本实施例实验所用DMEM培养基及新生牛血清购自Gibco公司；
本实施例实验所用Soimycin A购自美国Sigma公司；
5-氟尿嘧啶购自Sigma公司；
Annexin-PI凋亡试剂盒购自BD公司；
本实施例实验所用其它试剂为市售的分析纯试剂。
实施例1：5-FU对FoxM1基因敲除型小鼠生存率的影响
1.1实验方法：
FoxM1f1/f1Tie2Cre为FoxM1基因敲除型小鼠，FoxM1f1/f1为对照小鼠，Tie2Cre在不需要诱导物的情况下可以介导靶基因在内皮和造血系统的敲除，且敲除能够在整个造血过程中维持。分别选取一定数量的FoxM1f1/f1小鼠(共11只)及FoxM1f1/f1Tie2Cre小鼠(共12只)，通过腹腔注射5-氟尿嘧啶(150mg/kg)后，在6天及16天时再次注射，连续监测小鼠存活率。
1.2实验结果：
5-氟尿嘧啶是一种干扰DNA合成的广谱抗肿瘤药物，二次注射5-氟尿嘧啶后FoxM1f1/f1小鼠未出现死亡现象，三次注射5-氟尿嘧啶后FoxM1f1/f1小鼠存活率小幅降至90％。而FoxM1f1/f1Tie2Cre小鼠二次注射5-氟尿嘧啶后，在11天时开始出现急剧死亡现象，在三次注射前存活率已低于10％，P值小于0.0001，说明FoxM1部分介导了小鼠骨髓细胞的5-氟尿嘧啶耐药性，提示FoxM1可作为放化疗增敏剂的潜在靶点(参见图1)。
实施例2：Soimycin A预处理对髓性白血病细胞系放疗后凋亡率的影响
2.1实验方法：
分别接种K562及HL-60细胞系，分别利用0.5％DMSO及SoimycinA(0.5μM)预处理18小时，一组细胞利用总量为2Gy的放射性射线处理(0.5Gy/min)，另一组细胞静置，随后继续培养12小时。收集细胞后，Annexin V及DAPI双染色，流式细胞仪分析检测。
2.2实验结果：
DMSO预处理组K562及HL-60细胞经放疗后凋亡率分别由7％、8％升高至31％、41％。而Soimycin A预处理组K562及HL-60细胞经放疗后凋亡率分别由8％、8％升高至59％、76％。说明Soimycin A预处理可明显提高AML细胞系HL-60及CML细胞系K562放疗敏感性，且对HL-60细胞放疗增敏效果更为显著(参见图2)。
实施例3：Soimycin A预处理对髓性白血病小鼠化疗后生存率的影响
3.1实验方法：
一共20只NSGS受体小鼠进行2.5Gy的辐照，每个受体小鼠通过尾静脉注射0.2×106HL-60细胞，7天之后将受体小鼠分为两组各10个，进行5-氟尿嘧啶处理(150mg/kg)，其中一组注射5-氟尿嘧啶前24小时内注射两次DMSO作为对照组(12小时一次)，另外一组尾静脉注射两次Siomycin A(30mg/kg，12小时一次)，连续监测小鼠存活率。
3.2实验结果：
HL-60细胞移植构建的AML小鼠在接受5-氟尿嘧啶治疗前利用DMSO预处理，小鼠在23天时开始出现急剧死亡现象，在32天时全部死亡，而Soimycin A预处理组小鼠在25天时出现死亡现象，40天时尚有小鼠存活，p值小于0.05。说明Soimycin A可明显提高AML小鼠5-氟尿嘧啶治疗后存活率，证实Soimycin A具有AML小鼠化疗增敏的作用(参见图3)。
结论：FoxM1部分介导了小鼠骨髓细胞对5-氟尿嘧啶的耐药性，Siomycin A通过抑制FoxM1表达明显提高AML细胞系HL-60放疗敏感性，并提高HL-60细胞移植构建的AML小鼠化疗敏感性。
Claims (6)
1.Soimycin A在用于白血病放化疗增敏剂中的用途。
2.根据权利要求1所述的用途，其特征在于：所述白血病是急性髓性白血病。
3.根据权利要求2所述的用途，其特征在于：所述放化疗增敏剂通过抑制FoxM1的表达提高AML细胞系HL-60及CML细胞系K562放疗敏感性。
4.根据权利要求2所述的用途，其特征在于：所述放化疗增敏剂通过抑制FoxM1的表达提高HL-60细胞移植构建的AML小鼠化疗后存活率。
5.根据权利要求1或2所述的用途，其特征在于：所述放化疗增敏剂以注射剂或输液剂给药。
6.根据权利要求1或2所述的用途，其特征在于：利用SoimycinA制备的放化疗增敏剂用量为每公斤每12小时20～40mg。
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| WO2011127297A1 (en) * | 2010-04-07 | 2011-10-13 | The Board Of Trustees Of The University Of Illinois | Method of treating tumor resistant to herceptin or paclitaxel using foxm1 inhibitors and detecting same |
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