CN102552300B - 莫能菌素作为stat3信号特异性抑制剂的应用 - Google Patents
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Abstract
本发明提供了了一种莫能菌素的医药新用途——莫能菌素作为STAT3信号特异性抑制剂的应用。通过细胞模型筛选实验和Western-Blot检测实验证明，莫能菌素具有良好的STAT3抑制活性，并且能有效抑制STAT3所调控的相关基因的表达，从而可以有效抑制STAT3信号依赖的肿瘤细胞的生长，因此，以莫能菌素为活性成分，可用于制备基于STAT3为靶点的抗肿瘤药物：以莫能菌素为活性成分，采用药剂学上可接受的工艺和辅料，制备成各种剂型的抗肿瘤药物，如各种口服制剂和注射剂等。
Description
技术领域
本发明属于医药应用领域，涉及一种莫能菌素的医药用途，尤其涉及莫能菌素作为STAT3信号特异性抑制剂的应用。
背景技术
莫能菌素(Monensin)又称莫能霉素或瘤胃素，属一种聚醚类抗生素，在1967年首先由美国礼来公司Haney等人从肉桂地链霉菌（Streptomyces cinnamonensis）的发酵液中分离得到，是世界上使用最早、销量最大的广谱、高效的聚醚类抗生素，对牲畜多种主要球虫病均有效。其分子式为C36H62O11,分子量670.89，钠盐形式分子式为C36H62NaO11，分子量为692.85，结构式为：
莫能菌素具有控制瘤胃中挥发性脂肪酸比例，减少瘤胃中蛋白质降解，降低饲料干物质消耗，改善营养物质利用率和提高动物能量利用率等作用，其商品名为瘤胃素，是反刍动物中运用较广泛的饲料添加剂。
STAT3是至今研究非常透彻的与肿瘤相关的一系列信号传导分子中的重要成员之一，几乎参与了肿瘤发生的所有过程，包括了细胞的增殖、存活、血管生成、转移等关键步骤。结肠癌、乳腺癌、神经胶质瘤细胞、肺癌等细胞中，都有持续活化的 STAT3活性。白细胞介素6（IL-6） 参与的 STAT3 活化对早期结肠炎相关癌症的发生有重要关系(Grivennikov等人：Cancer Cell 2009)；同时 IL-6介导的 JAK-STAT 信号对前列腺癌细胞的增殖也有非常明显的促进作用(Lou等人：Prostate 2000)；由酪氨酸激酶参与的组成型 STAT3 活性促进了乳腺癌细胞的存活和增殖(Garcia等人：Oncogene 2001)；Src 诱导的STAT3活化对黑色素瘤的促进，以及STAT3 持续活化引起的VEGF的上调和肿瘤血管的生长也受到研究者们的关注(Niu等人：Oncogene 2002; Niu等人：Oncogene 2002; Wei等人：Oncogene 2003)；STAT3 及其调控的相关MMPs蛋白参与了肿瘤的浸润和转移(Xie等人：Cancer Res 2004; Xie等人：Oncogene 2006)；Kanda, N等人发现持续活化的 STAT3 能通过 Survivin 促进胃癌细胞的增殖(Kanda等人：Oncogene 2004)；此外 IL-6 信号参与了体内循环肿瘤细胞在原病灶部位的复发(Kim等人：J Gastroenterol Hepatol 2009)；STAT3 活性高是某些肿瘤预后差的重要原因之一(Kusaba等人：Oncol Rep 2006; Kim等人：Cell 2009)。最近的研究表明，像BMX非受体酪氨酸激酶参与的STAT3持续活化可在一定程度上维持神经胶质瘤干细胞样细胞的自我更新(Guryanova等人：Cancer Cell 2011)。研究还表明，在众多肿瘤细胞中，STAT3 和另外一个重要的肿瘤相关因子NF-kB还存在相互结合、共同作用促进肿瘤相关基因表达和肿瘤发生、发展的现象(Yang等人：Cancer Res 2005; Yang等人：Genes Dev 2007; Torres等人：Nat Cell Biol 2008; Lee等人：Cancer Cell 2009; Yu等人：Nat Rev Cancer 2009)。大量实验表明，通过特异性抑制 STAT3信号能在很大程度抑制肿瘤细胞的生存、增殖和迁移(Ni等人：Cancer Res 2000; Rahaman等人： Oncogene 2002; Leong等人：Proc Natl Acad Sci 2003; Barton等人：Mol Cancer Ther 2004; Darnell ：Nat Med 2005; Jing等人：Anticancer Drugs 2005; Kortylewski等人：Nat Med 2005; Xin等人：Cancer Res 2009)。目前以STAT3为靶点开发药物已成为公认的治疗某些肿瘤的重要方案，但是尚未出现理想的药物用于临床。
中国专利申请 201110423357.7公开了一种基于STAT3和NF-kB双信号通路为靶点的抗肿瘤药物筛选细胞模型及其构建和应用，在筛选相关抑制剂的过程中，发现莫能菌素能有效抑制所选用筛药模型中的荧光素酶活性，经过进一步验证发现，莫能菌素对STAT3信号有稳定的抑制效果，且在细胞水平具有高效低毒的抑制效果，能对STAT3信号依赖的肿瘤细胞具有较好的抑制作用。由于莫能菌素本身已用于畜牧生产，其副作用在可控范围之内，故作为STAT3信号抑制剂有很大潜力开发成药物治疗由于STAT3持续活化造成的肿瘤及相关疾病。
发明内容
本发明的目的是提供一种莫能菌素的医药新用途——莫能菌素作为STAT3信号特异性抑制剂的应用。
下面通过细胞实验来说明对莫能菌素（钠盐）（缩写成MoNa）对STAT3活性的抑制作用。
仪器：PerkinElmer Victor3 荧光/化学发光分析仪， Biorad 蛋白质电泳及转印系统，Biorad荧光定量PCR分析仪。
试剂：萤火虫荧光素酶测定试剂盒以及RNA反转录试剂盒购自Promega公司；IL-6购自Peprotech公司；莫能菌素钠购自 Sigma-Aldrich 公司；DMEM（高糖）及胎牛血清购自Hyclone（Thermo公司）；各种抗体购自Cell Signaling Technology公司；用于荧光定量PCR的Cyber Green购自Roche公司；总RNA提取试剂盒购自天根公司；MTT购自上海生工。
1、细胞模型筛选实验
利用一种基于STAT3和NF-kB双信号通路为靶点的抗肿瘤药物筛选细胞模型（药物筛选细胞模型的结构参见中国专利201110423357.7），用100 ml培养基接种于96孔板中，培养至细胞汇合度为30%~40%；使用10 μM的MoNa处理细胞，继续培养24 小时；在上述96孔板中加入50 ml稳定型荧光素酶底物，使用荧光/化学发光分析仪测定荧光强度并分析荧光抑制率。结果发现，MoNa能有效抑制所选用筛药模型中的荧光素酶活性；经过初步排查，发现MoNa对STAT3信号有稳定的抑制效果，抑制率稳定达到50%左右，且在细胞水平具有高效低毒的抑制效果。
2、不同浓度莫能菌素对STAT3活性的抑制实验
使用以STAT3为靶点的药物筛选细胞模型（药物筛选细胞模型的结构参见中国专利201110423004.7）验证莫能菌素对STAT3活性的抑制。分别用浓度为10 μM，8 μM，7μM，6 μM，5 μM，4 μM，3 μM，2 μM， 1 μM的MoNa处理筛药模型细胞株A549R，24小时后测定荧光；同时以MTT实验测定相应浓度下MoNa对该细胞的生长抑制作用。
以10,000 个/孔将细胞铺于96孔板，12h 后，将MoNa按上述浓度用完全培养基稀释，吸掉原来的培养基，加入含有MoNa的培养基，每个浓度n=6。MoNa母液用DMSO溶解，所用浓度培养基中DMSO含量不超过0.2%，并以不含MoNa的DMSO作为对照。24 h 后，3孔测定荧光素酶活性，3孔用MTT实验测定。测定荧光素酶活性时，每孔加入 50 μl稳定性荧光素酶底物，室温避光孵育10 min后，用荧光/化学发光分析仪测定荧光值。MTT试验中，加入 20μl 5 mg/ml MTT母液，37孵育2h后测定吸光值。
图1为不同浓度莫能菌素对STAT3活性的抑制率。图1的结果表明，在24 h内，从1~10μM浓度MoNa均对STAT3活性有较好的抑制作用，能达到40~50%的转录抑制活性，而对A549R细胞株本身并没有明显的毒性作用。
3、Western-Blot检测莫能菌素对STAT3活性的抑制
文献报道，在具有持续活化STAT3的细胞中，抑制STAT3将明显抑制这一类细胞的增殖后生长，人前列腺癌细胞DU145就是其中之一，抑制STAT3将有效抑制该细胞的增殖，故选用该细胞作为验证MoNa效果的细胞。
DU145细胞于10 cm 细胞培养皿中生长至汇合度为70%~80%，更换新鲜完全培养基，加入MoNa预处理4 h（10 μM），同时以加入等量DMSO作为对照，提取总蛋白，结果见图2。图2的结果表明，使用10 μM的MoNa处理DU145细胞4 h，能有效抑制DU145细胞中的STAT3活性。
4、莫能菌素对STAT3持续活化细胞的抑制增殖作用
使相同数目DU145细胞接种于96孔板至25%，换含有10 μM的MoNa的完全培养基处理细胞，以含有DMSO的完全培养基作为对照，分别处理24 h，48 h，72 h，加入20μl 5 mg/ml MTT母液，37孵育2h后测定吸光值（n=3）。检测结果见图3。图3 的结果显示，用MoNa长时间处理DU145细胞，发现MoNa能有效抑制DU145细胞的生长和增殖。
大量实验证明，MoNa对具有持续活化STAT3肿瘤细胞都具有明显的抑制增殖作用，如前列腺癌细胞、肺癌细胞、结肠癌细胞、宫颈癌细胞、乳腺癌细胞等。
5、Western-Blot对细胞因子IL-6介导的STAT3活性的抑制
为了进一步说明MoNa对STAT3活性的广泛性，使用STAT3本底活性低的细胞HEK293T，用IL-6额外刺激激活STAT3观察MoNa的效应。
Western-blot实验中，HEK293T细胞于10 cm 细胞培养皿中生长至汇合度为70%~80%，更换新鲜完全培养基，加入MoNa预处理30 min（10 μM），同时以加入等量DMSO作为对照，IL-6（250 ng/ml）处理2 h后提取总蛋白。图4为Western-Blot检测莫能菌素对细胞因子IL-6介导的STAT3活性的抑制。图4显示的结果发现，MoNa能有效抑制IL-6激活的STAT3，证明MoNa有比较普遍的使用范围。
6、荧光定量PCR检测莫能菌素对STAT3所调控下游基因的转录抑制
荧光定量PCR中HEK293T细胞于10 cm 细胞培养皿中生长至汇合度为70%-80%，更换新鲜完全培养基，加入MoNa预处理30 min（10 μM），同时以加入等量DMSO作为对照，4 h后提取总RNA，反转录后，做荧光定量分析。图5为荧光定量PCR分析莫能菌素对细胞因子IL-6介导的STAT3下游基因socs3的转录抑制。图5的结果表明，使用MoNa预处理的HEK293T细胞，IL-6诱导的STAT3下游基因socs3 mRNA水平明显低于未用MoNa预处理的细胞，证明MoNa可以抑制由STAT3调控的下游基因的转录。
综上所述，MoNa具有良好的STAT3抑制活性，并且能有效抑制STAT3所调控的相关基因的表达，从而可以有效抑制STAT3信号依赖的肿瘤细胞的生长，因此，以MoNa为活性成分，可用于制备基于STAT3为靶点的抗肿瘤药物：以MoNa为活性成分，采用药剂学上可接受的工艺和辅料，制备成各种剂型的抗肿瘤药物，如各种口服制剂和注射剂等。
附图说明
图1为不同浓度莫能菌素对STAT3活性的抑制
图2为Western-Blot检测莫能菌素对STAT3活性的抑制
图3为莫能菌素对肿瘤细胞的增殖抑制
图4为Western-Blot检测莫能菌素对细胞因子IL-6介导的STAT3活性的抑制
图5为荧光定量PCR分析莫能菌素对细胞因子IL-6介导的STAT3下游基因socs3的转录抑制。
具体实施方式
下面具体实施例对莫能菌素作为活性成分，在制备基于STAT3为靶点的抗肿瘤药物中的应用作进一步说明。
实施例1、莫能菌素片剂的制备
配方： 莫能菌素钠盐（纯度大于95%） 20.0g
填充剂 180.0g
崩解剂 10.0g
黏合剂 6.0g
润滑剂 3.0g
共计 200.0g
工艺：按制备片剂的常规工艺制成片剂1000片，每片含莫能菌素钠盐20mg。
实施例2：莫能菌素胶囊的制备
配方：莫能菌素钠盐（纯度大于95%） 50.0g
填充剂 85.0g
黏合剂 5.0g
润滑剂 10.0g
共计 200.0g
工艺：按制备胶囊剂的常规工艺制成1000粒，每粒胶囊含莫能菌素钠盐50mg。
实施例3、莫能菌素注射液的制备
配方：莫能菌素钠盐（纯度大于95%） 100.0g
枸橼酸 1.0g
枸橼酸钠 0.5g
氯化钠 18.0g
注射用水 2000ml
工艺：按制备注射剂的常规工艺操作，共制成2ml的注射剂1000支，每支莫能菌素钠盐100mg。
上述各实施例中的填充剂、崩解剂、 黏合剂、润滑剂等辅料均为药剂学上最常规的辅料。
Claims (1)
1.荧光定量PCR检测莫能菌素对STAT3所调控下游基因socs3的转录抑制的应用，其特征在于，该荧光定量PCR中HEK293T细胞于10 cm 细胞培养皿中生长至汇合度为70%-80%，更换新鲜完全培养基，加入10 μM莫能菌素预处理30 min，同时以加入等量DMSO作为对照，4 h后提取总RNA，反转录后，做荧光定量分析。
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