以STAT3为靶标的抗肿瘤药物高通量筛选模型的建立和应用

生物技术通报 ›› 2014, Vol. 0 ›› Issue (4): 187-192.

• 研究报告 • 上一篇

以STAT3为靶标的抗肿瘤药物高通量筛选模型的建立和应用

潘丽, 张宁, 牛国君, 孟晶, 刘祥, 杨诚

(1.天津科技大学生物工程学院，天津 300457；
2.南开大学药学院，天津 300071；
3.天津国际生物医药联合研究院，天津 300457；4.南开大学生命科学学院，天津 300071)

收稿日期:2014-01-13 出版日期:2014-04-29 发布日期:2014-04-29
作者简介:潘丽，女，硕士研究生，研究方向：高通量药物筛选；E-mail：panlippg@126.com
基金资助:
国家自然科学基金青年科学基金项目(31200641)

The Establishment and Application of a High Throughput Screening Model for Inhibitors of STAT3

Pan Li, Zhang Ning, Niu Guojun, Meng Jing, Liu Xiang, Yang Cheng

(1. College of Biotechnology，Tianjin University of Science and Technology，Tianjin 300457；
2. College of Pharmacy，Nankai University，Tianjin 300071；
3. Tianjin International Joint Academy of Biotechnology and Medicine，Tianjin 300457； 4. College of Life Sciences，Nankai University，Tianjin 300071)

Received:2014-01-13 Published:2014-04-29 Online:2014-04-29

摘要/Abstract

摘要： 旨在建立稳定可靠的以转导与转录激活子(STAT3)为靶标的抗肿瘤高通量筛选模型，应用该模型筛选潜在的抗癌药物。利用基因重组、蛋白表达纯化技术，获得STAT3目的蛋白，使用酶联免疫吸附法(ELISA)进行高通量药物筛选，将筛选出的抑制剂在细胞水平上利用MTT比色法测定化合物对癌细胞增殖的影响。结果显示，成功构建表达载体pET-28a-STAT3；所建立的模型稳定可行，可用于以STAT3为靶标的抗肿瘤药物的高通量筛选；用该模型对8 248个样品进行筛选，在500 μmol/L药物浓度下，化合物MDC6抑制率为92%，另外，进行IC₅₀值的测定时，分子水平上最低达到3.37 μmol/L，在细胞水平上可达到15.92 μmol/L。建立的高通量药物筛选模型，具有操作方便、成本低、结果稳定等特点，可用于STAT3抑制剂的大规模筛选。

关键词: STAT3, 靶点, 高通量模型, ELISA

Abstract: It was to establish a viable high-throughput drug screening model for discovering inhibitors of Signal Transducer and Activator of Transcription3, and applied the model to screen potential anti-cancer drugs. The STAT3 gene was amplified with PCR and cloned into the expression vector pET-28a. The recombinant STAT3 was over-expressed and purified, and then was used to bind with specific phosphotyrosine peptides. We established a high-throughput drug screening model based on ELISA to obtain some effective compounds. Further, we tested the effect of them on the growth ability and proliferation of the Hepatoma cells using MTT assay. Results showed that it was not only successfully constructed the expression vector pET-28a-STAT3, but also established a reliable and stable model for drug screening. Besides, 8 248 samples were screened, and a positive sample MDC6 was finally obtained. When the drug concentration was less than 500 μmol/L, the inhibition rate of MDC6 reached 92%. The minimum value of IC₅₀ was 3.37 μmol/L, while at the cellular level it was 15.92 μmol/L. In this work, we developed a repeatable and reliable assay for screening of STAT3 inhibitors.

Key words: STAT3, Target, High-throughput drug screening model, ELISA

潘丽, 张宁, 牛国君, 孟晶, 刘祥, 杨诚. 以STAT3为靶标的抗肿瘤药物高通量筛选模型的建立和应用[J]. 生物技术通报, 2014, 0(4): 187-192.

Pan Li, Zhang Ning, Niu Guojun, Meng Jing, Liu Xiang, Yang Cheng. The Establishment and Application of a High Throughput Screening Model for Inhibitors of STAT3[J]. Biotechnology Bulletin, 2014, 0(4): 187-192.

参考文献

[1] Yu H, Pardoll D, Jove R. STATs in cancer inflammation and immunity：a leading role for STAT3. Nature Reviews[J]. Cancer, 2009, 9(11)：798-809.
[2] Zhang X, Yue P, Page BD, et al. Orally bioavailable small-molecule inhibitor of transcription factor Stat3 regresses human breast and lung cancer xenografts[J]. Proceedings of the National Academy of Sciences, 2012, 109(24)：9623-9628.
[3] Spaccarotella E, Pellegrino E, Ferracin M, et al. STAT3-mediated activation of microRNA cluster 17~92 promotes proliferation and survival of ALK positive anaplastic large cell lymphoma[J]. Haematologica, 2014, 99(1)：116-124.
[4] Grivennikov S, Karin E, Terzic J, et al. IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer[J]. Cancer Cell, 2009, 15(2)：103-113.
[5] Iwamaru A, Szymanski S, Iwado E, et al. A novel inhibitor of the STAT3 pathway induces apoptosis in malignant glioma cells both in vitro and in vivo[J]. Oncogene, 2006, 26(17)：2435-2444.
[6] Frank DA. STAT3 as a central mediator of neoplastic cellular transformation[J]. Cancer Letters, 2007, 251(2)：199-210.
[7] Selvaraj BT, Frank N, Bender FL, et al. Local axonal function of ST-AT3 rescues axon degeneration in the pmn model of motoneuron dis-ease[J]. The Journal of Cell Biology, 2012, 199(3)：437-451.
[8] Hedvat M, Huszar D, Herrmann A, et al. The JAK2 inhibitor AZD1480 potently blocks Stat3 signaling and oncogenesis in solid tumors[J]. Cancer Cell, 2009, 16(6)：487-497.
[9] Lin L, Hutzen B, Zuo M, et al. Novel STAT3 phosphorylation inhibit-ors exhibit potent growth-suppressive activity in pancreatic and bre-ast cancer cells[J]. Cancer Research, 2010, 70(6)：2445-2454.
[10] Haan S, Hemmann U, Hassiepen U, et al. Characterization and binding specificity of the monomeric STAT3-SH2 domain[J]. The Journal of Biological Chemistry, 1999, 274(3)：1342-1348.
[11] Becker S, Groner B, Müller CW. Three-dimensional structure of the Stat3β homodimer bound to DNA[J]. Nature, 1998, 394(6689)：145-151.
[12] Schust J, Berg T. A high-throughput fluorescence polarization assay for signal transducer and activator of transcription 3[J]. Analytical Biochemistry, 2004, 330(1)：114-118.
[13] Mosmann T. Rapid colorimetric assay for cellular growth and survival：application to proliferation and cytotoxicity assays[J]. Journal of Immunological Methods, 1983, 65(1-2)：55-63.
[14] Song H, Wang R, Wang S, Lin J. A low-molecular-weight compound discovered through virtual database screening inhibits Stat3 function in breast cancer cells[J]. Proc Natl Acad Sci USA, 2005, 102(13)：4700-4705.
[15] Liby K, Voong N, Williams CR, et al. The synthetic triterpenoid CDDO-Imidazolide suppresses STAT phosphorylation and induces apoptosis in myeloma and lung cancer cells[J]. Clinical Cancer Research, 2006, 12(14)：4288-4293.
[16] Siddiquee K, Zhang S, Guida WC, et al. Selective chemical probe inhibitor of Stat3, identified through structure-based virtual screening, induces antitumor activity[J]. Proceedings of the National Academy of Sciences, 2007, 104(18)：7391-7396.

以STAT3为靶标的抗肿瘤药物高通量筛选模型的建立和应用

The Establishment and Application of a High Throughput Screening Model for Inhibitors of STAT3

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	周璐祺, 崔婷茹, 郝楠, 赵雨薇, 赵斌, 刘颖超. 化学蛋白质组学在天然产物分子靶标鉴定中的应用[J]. 生物技术通报, 2023, 39(9): 12-26.
[2]	沈俊强, 张莉萍, 于瑞明, 王永录, 潘丽, 刘霞, 刘新生. 猪嵴病毒结构蛋白VP0与VP1原核表达及间接ELISA方法的建立[J]. 生物技术通报, 2022, 38(10): 243-253.
[3]	吴玉苹, 周勇, 蒲娟, 李会, 章金刚, 朱艳平. 代谢组学在肿瘤药物靶点筛选中的应用进展[J]. 生物技术通报, 2022, 38(1): 311-318.
[4]	瞿欢, 李成, 陈汭, 廖艺杰, 曹三杰, 文翼平, 颜其贵, 黄小波. 猪δ冠状病毒S1-CTD的截短表达及间接ELISA抗体方法的建立[J]. 生物技术通报, 2021, 37(5): 273-280.
[5]	陈鹏. 活性天然产物蛋白靶点的快速筛选策略[J]. 生物技术通报, 2020, 36(11): 180-187.
[6]	盛树悦，陈悦，张兴梅，石玉生. 核糖开关及其在抗菌药物方面的研究进展[J]. 生物技术通报, 2017, 33(1): 114-119.
[7]	杨川,秦艺丹,胡潜,李强. 抗迟缓爱德华菌（Edwardsiella tarda）单克隆抗体的制备及双抗夹心ELISA检测方法的建立[J]. 生物技术通报, 2016, 32(7): 200-205.
[8]	刘鹏展, 黄绮玲, 何小维, 李文美, 张文琪, 张赛. NT-proBNP特异性单克隆抗体的制备及鉴定[J]. 生物技术通报, 2016, 32(6): 148-154.
[9]	尹珅,贺桂芳,赖方秾,谢凤云,马俊宇. CRISPR/Cas9系统的脱靶效应[J]. 生物技术通报, 2016, 32(3): 31-37.
[10]	李倩,王艳林,. EZH2作为抗肿瘤免疫治疗靶点研究进展[J]. 生物技术通报, 2015, 31(1): 29-32.
[11]	田园园叶星张莉莉. 草鱼IgM 的纯化、标记及检测方法的建立[J]. 生物技术通报, 2013, 0(4): 194-200.
[12]	李忠鹏, 于寒松, 胡耀辉, 时圣凤, 刘蕴慧, 段永杰, 李小宇, 王永志, 李启云. CP4-EPSPS 夹心ELISA 配对单克隆抗体的研制和生物学特性分析[J]. 生物技术通报, 2013, 0(4): 206-209.
[13]	夏武;贾岩龙;朱武凌;程娜;陈永凤;刘巨源;. RNAi沉默STAT3基因对人大细胞肺癌细胞增殖的影响[J]. , 2012, 0(06): 183-188.
[14]	周晓明;傅海媛;黄亚娟;侯利平;孙云波;原剑;杨保安;郑俊杰;甄蓓;肖汉族;貌盼勇;魏开华;. 多肽抗体检测原发性肝癌血清标志物ELISA方法的建立及应用[J]. , 2012, 0(05): 179-184.
[15]	杨磊;曹以诚;高强;葛洪;. 特异性表达siRNA的新型乙肝核酸药物的研究[J]. , 2012, 0(04): 181-185.