药学学报, 2019, 54(3): 475-481
引用本文:
钱亚芳, 杨波, 冯迪, 钱亿帆, 吴亚莉, 张姁, 谷满仓. “靶点+活性”快速发现苦蘵中靶向Hsp90抗胰腺癌有效成分的研究[J]. 药学学报, 2019, 54(3): 475-481.
QIAN Ya-fang, YANG Bo, FENG Di, QIAN Yi-fan, WU Ya-li, ZHANG Xu, GU Man-cang. The “target + activity” method for rapid discovery of active compounds targeting Hsp90 in pancreatic cancer cells from Physalis angulata L.[J]. Acta Pharmaceutica Sinica, 2019, 54(3): 475-481.

“靶点+活性”快速发现苦蘵中靶向Hsp90抗胰腺癌有效成分的研究
钱亚芳1, 杨波2, 冯迪2, 钱亿帆2, 吴亚莉2, 张姁2, 谷满仓2
1. 浙江中医药大学附属第一医院 (浙江省中医院), 浙江 杭州 310006;
2. 浙江中医药大学药学院, 浙江 杭州 310053
摘要:
本研究基于"靶点+活性"双重导向快速发现技术筛选苦蘵中靶向Hsp90蛋白杀伤胰腺癌细胞的活性单体。通过将体外抗肿瘤活性筛选技术、双萤光素酶报告基因技术与多元色谱分离技术相耦合,从中药苦蘵中筛选靶向Hsp90的活性单体。研究化合物抗胰腺癌细胞BXPC-3生长活性,初步探讨化合物抑制Hsp90分子机制。结果显示从苦蘵中分离得到醉茄内酯类化合物withanolide E(WE)与4β-hydroxywithanolide E(HWE)。MTT结果显示,WE与HWE处理BXPC-3细胞48 h的半数抑制率(IC50)分别为0.71±0.03和1.23±0.10 μmol·L-1;萤光素酶报告基因实验结果显示,WE与HWE可使细胞热休克元件活性增强12.5±3.4倍与28.1±3.4倍;两者均呈现量效与时效关系。分子机制研究提示,与DMSO组相比5 μmol·L-1 WE和HWE分别处理BXPC-3细胞48 h可诱导Hsp90二聚体蛋白表达上调6.5±1.3和11.8±2.0倍,Hsp90客户蛋白Akt表达下调至DMSO组的21.7%±2.8%和9.8%±1.4%;shRNA干扰Hsp90基因表达可阻断其抑制Akt表达与杀伤肿瘤细胞的作用。采用"靶点+活性"双重导向快速发现技术可从苦蘵中筛选得到靶向Hsp90蛋白杀伤胰腺癌细胞的活性单体WE与HWE;其分子机制可能与诱导胰腺癌细胞形成无活性Hsp90二聚体,进而抑制Hsp90客户蛋白Akt表达有关。
关键词:   
The “target + activity” method for rapid discovery of active compounds targeting Hsp90 in pancreatic cancer cells from Physalis angulata L.
QIAN Ya-fang1, YANG Bo2, FENG Di2, QIAN Yi-fan2, WU Ya-li2, ZHANG Xu2, GU Man-cang2
1. The First Affiliated Hospital of Zhejiang Chinese Medical University(Zhejiang Provincial Hospital of Traditional Chinese Medicine), Hangzhou 310006, China;
2. School of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou 310053, China
Abstract:
The purpose of this study was to select the active compounds targeting Hsp90 protein in pancreatic cancer cells through a new dual "target + activity" rapid discovery technique. We combined an in vitro anti-cancer activity screening method with a dual-luciferase reporter gene and multi-chromatography separation technology, for rapid discovery of potential Hsp90 inhibitors from the Chinese herbal medicine Physalis angulata L. The anti-proliferation activity of those compounds was assessed in pancreatic cancer cell line BxPC-3 by MTT assays. The molecular mechanisms of Hsp90 inhibition were explored by Western blot and shRNA knockdown assays. As a result, two withanolides, withanolide E (WE) and 4β-hydroxywithanolide E (HWE), were identified from Physalis angulata L. The half maximal inhibitory concentration (IC50) of WE and HWE were 0.71±0.03 and 1.23±0.10 μmol·L-1 for the growth of BxPC-3 cells in 48 h. Luciferase reporter assay demonstrated that WE and HWE significantly induced heat shock element (HSE) activity in a dose-and time-dependent manner. The molecular mechanism study showed that after exposing to 5 μmol·L-1 WE or HWE for 48 h, the aggregation of Hsp90 dimer was upregulated to 6.5±1.3 and 11.8±2.0 fold, while the expression of Hsp90 client protein Akt was downregulated to 21.7%±2.8% and 9.8%±1.4% of the control group. Moreover, the Hsp90 inhibitory activity of WE or HWE was canceled by shRNA mediated Hsp90 knockdown. Overall, based on the dual "target + active" rapid discovery technique, two new Hsp90 inhibitors WE and HWE were found from Physalis angulata L. The Hsp90 inhibitory mechanism of WE and HWE may be mediated by induction of Hsp90 aggregate dimer and inhibition of Hsp90 client protein Akt expression.
Key words:   
收稿日期: 2018-09-30
DOI: 10.16438/j.0513-4870.2018-0891
基金项目: 国家自然科学基金资助项目(81673607,81303235,81774011);浙江省自然科学基金资助项目(Y19H280009,Y17H280023);湖州市科技计划公益类研究(2018GZ24).
通讯作者: 谷满仓
Email: gmancang@zcmu.edu
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参考文献:
[1] Zhang SR, Jing W, Liu L, et al. New progress in basic research and diagnosis and treatment of pancreatic cancer in 2017[J]. Chin Oncol (中国癌症杂志), 2018, 28:1-10.
[2] Shrestha L, Bolaender A, Patel HJ, et al. Heat shock protein (HSP) drug discovery and development:targeting heat shock proteins in disease[J]. Curr Top Med Chem, 2016, 16:2753-2764.
[3] Ding QH, Cheng Y, Chen WP, et al. Celastrol, an inhibitor of heat shock protein 90beta potently suppresses the expression of matrix metalloproteinases, inducible nitric oxide synthase and cyclooxygenase-2 in primary human osteoarthritic chondrocytes[J]. Eur J Pharmacol, 2013, 708:1-7.
[4] Fan YJ, Liu Y, Zhang L, et al. C0818, a novel curcumin derivative, interacts with Hsp90 and inhibits Hsp90 ATPase activity[J]. Act Pharm Sin B, 2017, 7:91-96.
[5] Fang JJ, Zheng XL, Xia H, et al. Study on the chemical constituents and cytotoxic activity of Physalis angulata L.[J]. Chin Trad Herb Drugs (中草药), 2017, 48:1080-1086.
[6] Hu YJ, Wang YQ, Wang YP, et al. Discussion on the selection, compatibility and quality control of the medicinal componens in traditional Chinese medicine[J]. Chin Trad Herb Drugs (中草药), 2016, 47:2965-2971.
[7] Pang WQ, Gao L, Dou YY, et al. Screening of natural small molecule IDO-1 inibitors with anti-tumor role[J]. Acta Pharm Sin (药学学报), 2017, 52:1416-1423.
[8] Joshi P, Misra L, Siddique AA, et al. Epoxide group relationship with cytotoxicity in withanolide derivatives from Withania somnifera[J]. Steroids, 2014, 79:19-27.
[9] Maldonado E, Alvarado VE, Torres FR, et al. Androstane and withanolides from Physalis cinerascens[J]. Planta Med, 2005, 71:548-553.
[10] Quan NH, Kang YJ, Li DH, et al. Research progress of high-throughput screening technology for active ingredients of Chinese herbal medicine[J]. J Analy Sci (分析科学学报), 2017, 33:741-746.
[11] Yang B, Shen JW, Zhou DH, et al. Precise discovery of a STAT3 inhibitor from Eupatorium lindleyanum and evaluation of its activity of anti-triple-negative breast cancer[J]. Nat Prod Res, 2017, 31:1-9.
[12] Zhu XH, Ando J, Takagi M, et al. Six new withanolide-type steroids from the leaves of Solanum cilistum[J]. Chem Pharm Bull (Tokyo), 2001, 49:161-164.
[13] Zhang H, Samadi AK, Cohen MS, et al. Anti-proliferative withanolides from the Solanaceae:a structure-activity study[J]. Pure Appl Chem, 2012, 84:1351-1367.
[14] Henrich CJ, Brooks AD, Erickson KL, et al. Withanolide E sensitizes renal carcinoma cells to TRAIL-induced apoptosis by increasing cFLIP degradation[J]. Cell Death Dis, 2015, 6:e1666.
[15] Park EJ, Sang-Ngern M, Chang LC, et al. Induction of cell cycle arrest and apoptosis with downregulation of Hsp90 client proteins and histone modification by 4beta-hydroxywithanolide E isolated from Physalis peruviana[J]. Mol Nutr Food Res, 2016, 60:1482-1500.
[16] Nardai G, Sass B, Eber J, et al. Reactive cysteines of the 90-kDa heat shock protein, Hsp90[J]. Arch Biochem Biophys, 2000, 384:59-67.
[17] Chen WY, Chang FR, Huang ZY, et al. Tubocapsenolide A, a novel withanolide, inhibits proliferation and induces apoptosis in MDA-MB-231 cells by thiol oxidation of heat shock proteins[J]. J Biol Chem, 2008, 283:17184-17193.
[18] Yu Y, Hamza A, Zhang T, et al. Withaferin A targets heat shock protein 90 in pancreatic cancer cells[J]. Biochem Pharmacol, 2010, 79:542-551.
[19] Sarkar S, Dutta D, Samanta SK, et al. Oxidative inhibition of Hsp90 disrupts the super-chaperone complex and attenuates pancreatic adenocarcinoma in vitro and in vivo[J]. Int J Cancer, 2013, 132:695-706.
[20] Xue NN, Jin J, Chen XG. Co-chaperones:regulated action in conformational functions of HSP90 and their actions in cancer[J]. Acta Pharm Sin (药学学报), 2017, 52:1085-1090.
[21] Belalcazar A, Shaib WL, Farren MR, et al. Inhibiting heat shock protein 90 and the ubiquitin-proteasome pathway impairs metabolic homeostasis and leads to cell death in human pancreatic cancer cells[J]. Cancer, 2017, 123:4924-4933.
[22] Yamada-Kanazawa S, Kajihara I, Fukushima S, et al. Inhibition of heat shock protein 90 exerts an antitumour effect in angiosarcoma:involvement of the vascular endothelial growth factor signalling pathway[J]. Br J Dermatol, 2017, 177:456-469.