CN100413493C - 棉酚及其衍生物在制备治疗骨髓瘤药物中的应用 - Google Patents
棉酚及其衍生物在制备治疗骨髓瘤药物中的应用 Download PDFInfo
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Abstract
本发明涉及棉酚及其衍生物在制备治疗白血病/骨髓瘤药物中的应用。所述的棉酚的结构式如下：所述的棉酚衍生物为醋酸棉酚或甲酸棉酚；本发明的优益之处在于：棉酚及其衍生物能有效杀伤具有抗药性的白血病/骨髓瘤癌症细胞，而对正常细胞没有杀伤能力，并且特别适用于耐常规化疗药物的癌症病人和癌症复发病例。
Description
技术领域
本发明属于药物领域，特别涉及一种棉酚及其衍生物在制备治疗白血病/骨髓瘤药物中的应用。
技术背景
目前临床癌症的主要治疗方法是化学药物治疗，即大剂量DNA损伤剂的应用。这种方法副作用很大，因为化疗剂在杀伤癌症细胞的同时也大量破坏正常细胞。不仅如此，肿瘤细胞在治疗过程中会产生抗药性，导致癌症的复发。因此，寻找能选择性杀伤癌症细胞，特别是耐常规化疗药物的肿瘤细胞已经成为药物研发的热点。
癌症抗药性的原因较多。最新的研究表明，癌症的抗药性与癌症细胞的凋亡异常有关，其中Bcl-2家族蛋白在细胞凋亡的精细调控中起着非常重要的作用。该家族蛋白包含两类功能相反的蛋白：一类是抗凋亡蛋白，包括Bcl-2和Bcl-xL等十余个成员；另一类是促凋亡蛋白，包括Bax、Bak、Bid和Bad等十余个成员。抗凋亡蛋白如Bcl-2和Bcl-xL通过其BH3结构域能与一定数目的促凋亡蛋白如Bak、Bid、Bad、Bax形成异二聚体，它们的相互作用调节着细胞的凋亡或生存。临床研究发现，细胞内Bcl-2/Bcl-xL蛋白表达异常与多种癌症的发生有关，许多肿瘤细胞高表达Bcl-2/Bcl-xL蛋白，另外，细胞内Bcl-2/Bcl-xL表达的增多也与癌细胞抵抗广谱化疗药物以及放疗相关。因此，Bcl-2/Bcl-xL已经是目前国际上开发抗癌药物的一个新的药物靶点，其研发工作集中在如何抑制Bcl-2/Bcl-xL活性或降低其表达水平，来诱导癌症细胞的凋亡，从而达到治疗的目的。
棉酚及其衍生物是一种具有广泛药理作用的多元酚类化合物，大量的研究表明，棉酚及其衍生物具有抗生育、抗炎症、抗病毒、抗寄生虫的作用。另外，棉酚及其衍生物还具有杀伤某些肿瘤细胞的作用，如中国专利ZL97105968.3、美国专利US6114397A中所述，目前国内外对棉酚及其衍生物杀伤肿瘤细胞活性的研究主要集中在男女生殖系统的肿瘤，如宫颈癌、子宫肌瘤、卵巢癌、乳腺癌、肾上腺皮质癌、前列腺癌，睾丸癌。但对棉酚及其衍生物在治疗白血病/骨髓瘤中的应用，特别是有效杀伤BcL-2高表达的白血病细胞的研究，尚未见报道以及专利的申请。
发明内容
本发明的目的在于提供棉酚及其衍生物在制备治疗白血病/骨髓瘤药物中的应用.
本发明所述的棉酚的结构式如下：
其衍生物为醋酸棉酚或甲酸棉酚.
本发明是通过建立细胞凋亡的技术平台，从中药库中和化学药库中近10000个化合物中筛选出棉酚及其衍生物，并通过一系列实验证明其为具有细胞通透性、非免疫原性的小分子化合物，能够抑制肿瘤细胞Bcl-2/Bcl-xL的活性，诱导肿瘤细胞凋亡，从而达到肿瘤治疗的目的。
本发明的优益之处在于：棉酚及其衍生物能有效杀伤具有抗药性的白血病/骨髓瘤癌症细胞，而对正常细胞没有杀伤能力，并且特别适用于耐常规化疗药物的癌症病人和癌症复发病例.
附图说明
图1是肿瘤细胞的生长与棉酚浓度的关系图；
其中：“-◆-”代表骨髓瘤细胞IM9/BcL-2，
“-■-”代表骨髓瘤细胞U266，
“-▲-”代表白血病细胞HL60.
图2是肿瘤细胞的生长与醋酸棉酚浓度的关系图；
其中：“-◆-”代表骨髓瘤细胞IM9/BcL-2，
“-■-”代表骨髓瘤细胞U266，
“-▲-”代表白血病细胞HL60.
图3是凋亡细胞与棉酚浓度的关系图；
其中：“-◆-”代表骨髓瘤细胞IM9/BcL-2，
“-■-”代表骨髓瘤细胞U266，
“-▲-”代表白血病细胞HL60。
图4是棉酚和醋酸棉酚处理的IM9/Bcl-2细胞线粒体膜势能与处理时间的关系图；
其中：“-◆-”代表棉酚，
“-■-”代表醋酸棉酚。
图5是棉酚引起的鼠肝脏线粒体细胞色素C的释放与棉酚浓度的关系；
图6是特异性抑制剂CsA和Bcl-xL能够完全抑制棉酚诱导的细胞色素C的释放；
图7是棉酚诱导细胞凋亡过程中Caspase-3裂解为17kD的小片断。
具体实施方式
实施例1、新的Bcl-2/Bcl-xL小分子抑制剂棉酚及其衍生物醋酸棉酚的鉴定
建立竞争性与Bcl-xL结合的体外实验模型。将人重组的Bcl-xL蛋白包被在96孔板上，加入FITC标记的人Bid-BH3多肽，其氨基酸序列为Ile Ile Arg Asn Ile Ala ArgHis Leu Ala Gln Val Gly Asp Ser Met Asp Arg，再加入中国医学科学院药物研究所国家药物筛选中心提供的10000种不同的待选小分子化合物，进行反应，使用荧光分光光度计POLAR STAR检测96孔板的荧光变化，如果小分子化合物能够抑制FITC-Bid-BH3与Bcl-xL的结合，则FITC-Bid-BH3荧光强度明显减弱，经过初步的筛选，15种小分子化合物能够强有力地抑制FITC-Bid-BH3与Bcl-xL的结合，其中棉酚及醋酸棉酚的的抑制活性最强，IC50为7.2μM，因此筛选出做进一步做细胞水平的研究。
将棉酚和醋酸棉酚与Bcl-2高表达的人多发性骨髓瘤IM9细胞(转染人Bcl-2cDNA的稳定表达细胞系IM9/BcL-2)在37℃，5％CO2培养箱中孵育48小时，加入MTS试剂，继续培养2-3小时，ELISA读板机在490nm波长下分析不同浓度的药物对细胞的生长抑制情况。结果表明，小分子化合物棉酚及其衍生物醋酸棉酚具有细胞膜通透性，能够拮抗Bcl-2的生物学功能，有效地抑制IM9/Bcl-2细胞的增殖。这进一步证明棉酚和醋酸棉酚就是我们要筛的新的Bcl-2/Bcl-xL小分子抑制剂。根据图1所示的肿瘤细胞的生长与棉酚浓度的关系图，说明棉酚能够有效抑制Bcl-2高表达的IM9/Bcl-2细胞、人骨髓瘤细胞U266和白血病细胞HL60细胞的增殖，并呈浓度依赖性关系。根据图2所示的肿瘤细胞的生长与醋酸棉酚浓度的关系图，说明醋酸棉酚也能够有效抑制Bcl-2高表达的IM9/Bcl-2细胞、人骨髓瘤细胞U266和白血病细胞HL60细胞的增殖，并呈浓度依赖性关系。上述研究表明，棉酚和醋酸棉酚能够抑制人白血病/骨髓瘤细胞的增殖，特别是能够特异地抑制Bcl-2/Bcl-xL高表达的骨髓瘤细胞生长.
实施例2、棉酚及其衍生物醋酸棉酚诱导Bcl-2高表达的人骨髓瘤细胞及其它白血病细胞凋亡的实验
接着我们研究了棉酚和醋酸棉酚能否诱导白血病细胞凋亡。我们应用目前国内外普遍使用的AnnexinV染色这一检测指标，来定量检测棉酚和醋酸棉酚诱导细胞凋亡的情况。将不同种类的人骨髓瘤细胞和白血病细胞接种在24孔板中，加入不同浓度的棉酚或醋酸棉酚处理48小时，PBS洗两次，加入FITC标记的AnnexinV，利用流式细胞计(FACSCalibur)检测药物对肿瘤细胞的杀伤效应。由图3凋亡细胞与棉酚浓度的关系图所示，棉酚在48小时内能有效地诱导IM9/Bcl-2骨髓瘤细胞、U266细胞和HL60细胞的凋亡，并呈现浓度依赖性关系。棉酚浓度在20μM时，80％以上的细胞出现凋亡。从表1的研究结果可以看出，棉酚的衍生物醋酸棉酚也能杀伤Bcl-2高表达的骨髓瘤细胞和其它白血病细胞，并呈现浓度依赖性关系.另外，临床常规应用的抗癌药物如VP16，顺铂等不能诱导Bcl-2高表达的骨髓瘤细胞凋亡。将棉酚和醋酸棉酚与分离的正常人的外周血白细胞孵育，也未出现细胞毒效应。上述研究表明，棉酚及其衍生物醋酸棉酚对白血病/骨髓瘤细胞具有特异杀伤效应，尤其是能够杀伤Bcl-2/Bcl-xL高表达的耐常规药物的肿瘤细胞，而对正常人外周血白细胞无明显杀伤作用。
表1醋酸棉酚诱导细胞凋亡与其浓度依赖关系
*凋亡细胞百分率， 均数±标准差
实施例3、棉酚及其衍生物醋酸棉酚诱导细胞凋亡是经过线粒体途径
(1)棉酚及其衍生物醋酸棉酚能够引起细胞线粒体膜势能的下降
将棉酚及其衍生物醋酸棉酚处理的IM9/Bcl-2细胞与荧光探针3，3’-dihexyloxacarbocynin iodide[DiOC6(3)]一起孵育，通过流式细胞术分析细胞荧光强度的强弱，检测线粒体膜势能的变化，如图3所示，棉酚及其衍生物醋酸棉酚处理的IM9/Bcl-2细胞线粒体膜势能与处理时间的关系图表明，两者均能导致线粒体膜势能(Δψm)的下降，且呈时间依赖性关系，处理16小时后线粒体膜势能下降了80％。
通过差速离心法分离细胞内线粒体，进行的无细胞体系实验进一步证明，棉酚能快速引起线粒体膜势能的下降，而重组的人Bcl-xL蛋白能够抑制棉酚引起的线粒体膜势能的下降，上述研究结果表明：棉酚是具有线粒体毒性的药物，其作用的靶点在线粒体外膜蛋白Bcl-xL上。
(2)棉酚能够引起线粒体细胞色素C的释放
将健康的Balb/C鼠断颈处死，迅速取出肝脏，匀浆，应用差速离心法提取线粒体，Western Blotting免疫印迹实验检测药物诱导线粒体细胞色素C释放的情况。实验结果见图5，棉酚引起的鼠肝脏线粒体细胞色素C的释放与棉酚浓度的关系表明棉酚能够引起线粒体细胞色素C的释放，并呈现浓度依赖性关系。
由图6可见，PTP孔开放的特异性抑制剂(CsA)和Bcl-xL，能够完全阻断棉酚引起的细胞色素C释放，进一步说明棉酚是直接作用于线粒体的细胞毒性药物，其作用靶点是线粒体外膜蛋白Bcl-xL。
实施例4、棉酚引起的细胞凋亡部分是由Caspase介导的
将人骨髓瘤细胞系IM9-Bcl-2细胞接种在24孔板中，加入20μM棉酚处理不同时间，检测药物诱导肿瘤细胞凋亡过程中Caspase的活化情况，以及Caspase抑制剂z-VAD-fmk对棉酚诱导细胞凋亡的抑制情况。结果显示：棉酚处理IM9/BcL-2细胞4小时，Caspase-3的活性明显升高，同时出现Caspase-3酶原的裂解，裂解为17kD的小片断，见图7，说明棉酚诱导的细胞凋亡是有Caspase-3介导的。Caspase广谱抑制剂z-VAD-fmk只能部分抑制IM9/Bcl-2细胞的凋亡，抑制率为30％～40％。研究表明，棉酚引起的Bcl-2/Bcl-xL高表达细胞的凋亡存在Caspase-依赖和Caspase-非依赖的两种形式。
利用细胞凋亡的技术平台和理论，采用高通量的大规模筛选检测体系，从中药和化学合成药物库中鉴定出了具有细胞通透性、非免疫原性的棉酚及其衍生物醋酸棉酚，它能特异地结合细胞线粒体外膜蛋白Bcl-xL，拮抗Bcl-2/Bcl-xL的活性，促使Bcl-2家族蛋白形成的膜孔道开放，细胞色素C释放，随后激活Caspase-9以及凋亡执行分子caspase-3，最后导致细胞的凋亡。由于棉酚及其衍生物醋酸棉酚能够有效地杀伤Bcl-2高表达的骨髓瘤/白血病细胞，而对人正常的白细胞无毒副作用。因此我们相信，棉酚和其衍生物是潜在的具有发展前景的抗白血病新药，特别是对常规化疗药物有耐药性的骨髓瘤和白血病细胞的治疗，具有广阔的应用前景。
甲酸棉酚杀肿瘤细胞活性实验与棉酚和醋酸棉酚一样。
研究表明，醋酸棉酚能够抑制白血病/骨髓瘤细胞的生长，诱导高表达Bcl-2分子的IM9骨髓瘤细胞凋亡，而对正常人的外周血白细胞无明显毒副作用，甲酸棉酚的杀肿瘤细胞活性呈现浓度依赖性和时间依赖性。在诱导细胞凋亡的信号传递方面，甲酸棉酚能够引起线粒体膜势能的下降，线粒体内细胞色素c释放，随后引起caspase-9以及凋亡执行分子caspase-3的活化，后者裂解胞浆和胞核的底物，最后导致细胞凋亡.因此棉酚及其衍生物醋酸棉酚和甲酸棉酚均是潜在的抗白血病/骨髓瘤药物。
Claims (1)
1. 棉酚及其衍生物在制备治疗骨髓瘤药物中的应用，所述的棉酚的结构式如下：
所述的棉酚衍生物为醋酸棉酚或甲酸棉酚。
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| CN1144089A (zh) * | 1995-09-01 | 1997-03-05 | 上海古康生物实业有限公司 | 水溶性棉酚制剂及其制备方法 |
| CN1204510A (zh) * | 1997-07-09 | 1999-01-13 | 辛国芳 | 以棉酚或(和)其衍生物为有效成分的药栓及用途 |
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| CN1144089A (zh) * | 1995-09-01 | 1997-03-05 | 上海古康生物实业有限公司 | 水溶性棉酚制剂及其制备方法 |
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