|Place of Conferral||北京|
|Keyword||对叶大戟 假白榄烷型大环二萜 肿瘤多药耐药逆转 P-gp蛋白调节剂|
肿瘤多药耐药(multidrug resistance，MDR)，指一种药物作用于肿瘤使之产生耐药性后，该肿瘤对从未接触、结构无关、靶点不同、机制各异的多种抗肿瘤药也具有交叉耐药性的现象。其最主要的作用机制之一是ABC家族转运蛋白(目前研究最广泛、最深入的为ABCB1基因编码的P-gp蛋白)的过表达，导致药物外排增加，形成耐药性。目前从天然产物中寻找能够靶向ABC转运体蛋白的调节剂已经成为全球先导化合物开发最具潜力的方法之一。大戟属植物中的一类特征性成分——大环二萜，因具有较好的靶向性与突出的MDR逆转活性而受到广泛关注。对叶大戟（Euphorbia sororia A. Schrenk）是大戟科（Euphorbiaceae）大戟属（Euphorbia）一年生草本植物，其果实作为传统维吾尔药材，具有利水消肿、镇咳平喘、燥湿开胃、健脑益智的作用。目前已报道的从对叶大戟全草和果实中分离得到的17个大环二萜均为假白榄烷（jatrophane）型骨架结构，部分化合物被证实具有不同程度的肿瘤MDR逆转活性。因此，本研究以体外肿瘤MDR逆转活性为导向针对对叶大戟果实中的大环二萜类成分进行进一步研究，以期发现更多结构新颖的活性成分。本研究主要包括以下两个部分：1. 对叶大戟果实中的大环二萜类成分研究通过“活性跟踪”并利用现代色谱分离技术和结构鉴定技术对对叶大戟果实的化学成分进行了研究。从其95%乙醇提取物中分离并鉴定出15个单体化合物，均为假白榄烷（jatrophane）型大环二萜（以ES系列顺次命名）。其中包括5个新化合物：euphosorophane A~E (ES-18 ~ ES-22)，化合物ES-23为首次从该种植物中分离得到，其余化合物均为已知。化合物ES-18和ES-21的绝对构型通过X-射线单晶衍射方法确定。同时，在分离过程中结合UPLC色谱分析法与活性筛选，得到了一个富含多数ES系列化合物且活性较理想的组分。2. 对叶大戟果实中活性成分逆转MDR作用及机理的研究考察了15个化合物的细胞毒及MDR逆转活性，发现这一系列的化合物对MCF-7和MCF-7/ADR细胞均有较弱的细胞毒作用，并且能够从不同程度逆转MDR。其中有8个化合物的活性优于阳性对照维拉帕米（VRP）。结合这一活性结果，对假白榄烷型二萜骨架上C-5及C-14位取代基性质对MDR逆转作用的影响进行了构效关系总结：（1）C-5位上不同酰氧基取代，将其按对活性有利程度排序为：-OMeBu > -OBz > -OPr > -OiBu；（2）C-14位含有芳香环取代对活性更有利，乙酰氧基或羰基取代对活性影响均不大。通过考察体外半最大效应浓度（EC50）与体外治疗指数（TI）这两个指标，从活性较优的8个化合物中挑选出一个最具研究潜力的化合物ES-18 (EC50 = 92.68 ± 18.28 nM，TI = 1059.56)。针对目前研究最广泛且深入的P-gp蛋白介导的MDR机制，运用特异性高表达P-gp的人乳腺癌阿霉素耐药细胞MCF-7/ADR模型，研究ES-18逆转耐药的机理：首先通过MTT法观察到ES-18除能够逆转阿霉素（DOX）耐药外，还能有效逆转其他结构不同作用机制各异但同为P-gp底物的抗肿瘤药——多西他赛和长春新碱的耐药性。结合荧光显微镜观察及流式细胞法，发现ES-18能够呈一定浓度依赖性地增加P-gp特异性底物Rh123与DOX的积累量，且同浓度下作用优于VRP。通过western blot分析发现ES-18不影响P-gp蛋白的表达量，而在检测ES-18对Pgp-ATPase活性时观察到ES-18能够充当P-gp的底物激活ATPase酶活性，结果显示ES-18可通过直接抑制P-gp功能而逆转耐药。通过动力学考察中Lineweaver-Burk与Dixon两种双倒数作图法，发现ES-18是P-gp底物DOX的竞争性抑制剂，并且ES-18对P-gp具有更高的亲和性(Ki = 0.49 ~ 0.50 μM)。通过分子对接方法进一步模拟、确认了ES-18与其靶蛋白P-gp的作用。综上所述：化合物ES-18作为充当P-gp底物的一类抗肿瘤药的竞争性抑制剂发挥其多药耐药逆转作用，是一种比VRP更为有效的靶向P-gp的MDR逆转剂。
Multidrug resistance (MDR), which is characterized by cross-resistance to a broad spectrum of unrelated drugs that differ widely with respect to molecular structure and target specificity, is becoming a major reason why cancer treatments may fail. The overexpression of P-glycoprotein (P-gp) is the most common mechanism of MDR. P-gp, a plasma membrane-spanning transport protein in drug resistant cancer cells, can recognize chemotherapeutic drugs as substrates and extrude them out of cells using the energy of ATP hydrolysis, leading to ineffective intracellular drug concentrations and poor clinical outcomes following chemotherapy. Apart from synthetic compounds, the development of natural products is one of the most potential approaches of global drug discovery. Many nontoxic natural products including various extracts and active components have already been investigated to overcome MDR with some success. In particular, macrocyclic diterpenes, which were isolated from different Euphorbia species with a broad structural diversity, especially for jatrophanes and lathyranes, showed promising efficiency to be able to reverse P-gp-mediated MDR.Euphorbia. sororia A. Schrenk, an annual herb, is mainly found in northwest China and Central Asia. Fructus of E. sororia, a traditional Uyghur medicine, has been used to cure abdominal pain, abdominal distention, skin disease, and paralysis. Previous phytochemical investigations on this plant yielded seventeen jatrophane diterpenoids, some of them exerted different potent of MDR-reversal activity.Herein, we intended to search for a series of structurally related jatrophane diterpenoids for the purpose of developing novel jatrophane MDR modulators with enhanced safety and MDR reversal effect. The present study included two parts: 1. Jatrophane diterpenoids in fructus of E. sororia.On the basis of bioactivity guided isolation, five new jatrophane diterpenoids, named euphosorophane A~E (ES-18~ES-22), together with ten known jatrophane diterpenoids were isolated from the fructus of E. sororia and their structures were elucidated by extensive spectroscopic analysis. The absolute configurations of compounds ES-18 and ES-21 were confirmed by X-ray crystallographic analysis. In addition, followed by a bioactivity-guided isolation, one fraction was found to be an effective MDR-reversal agent. 2. Jatrophane diterpenoids from E. sororia as potent modulators against P-gp-based multidrug resistance.We evaluated these 15 jatrophane diterpenoids’ effects on MDR reversal activity, cytotoxicity against cancer cells and normal cells. After preliminary biological evaluations, some supplementary notes of SAR in jatrophane explained that the activity order of the ester groups at C-5 is 2-methylbutanoyloxy > benzoyloxy > propionyloxy > isobutanoyloxy. In addition, the presence of an aromatic ester group (benzoyl) at C-14 could increase the modulation potency in comparison to a substituent group of carbonyl or acetoxyl. The biological evaluation in vitro demonstrated that a few compounds exhibited potent MDR reversal activity. In particular, compound ES-18 exhibited outstanding MDR reversal activity (IC50 = 2.65 ± 0.33 μM) compared to the other compounds with a low EC50 value (92.68 ± 18.28 nM) in the MCF-7/ADR cell lines overexpressing P-gp. The significant advantage of ES-18 includes its high survival potency toward normal cell line HEK293 (IC50 = 98.20 ± 1.59 μM) and its high therapeutic index (1059.56). Compound ES-18 also exhibited reversal effects on the other cross-resistant chemotherapeutic drugs that have different structures such as docetaxel and vincristine. Compound ES-18 exhibited remarkable potency in inhibiting the P-gp-mediated drug efflux of Rhodamine123 or doxorubicin (DOX) in the MCF-7/ADR cells. Results of the western blotting assay suggested that MDR reversal by ES-18 was not due to a decrease in the protein expression of P-gp. The preliminary results of the Pgp-ATPase activity assay indicated that ES-18 could stimulate Pgp-ATPase activity by binding to the substrate recognition site. Kinetic characterization suggested that ES-18 may well be a competitive inhibitor of DOX with about 5.84- to 5.88- fold lower Ki values than the average Ki of verapamil (VRP), suggesting that ES-18 possessed a high binding affinity toward the DOX recognition site of P-gp. This was almost in accordance with our observation that 4-fold more of VRP, compared with ES-18, was needed to fully restore the DOX accumulation in the MCF-7/ADR cells to the level of the parental MCF-7 cells. This result indicated that the modulation mechanism of ES-18 was dependent on its direct inhibition effect of P-gp-mediated DOX efflux, thus increasing the intracellular DOX accumulation and finally restoring chemosensitivity of MCF-7/ADR to DOX. Then we performed a virtual docking experiment to prove ES-18 has the same effect as VRP for its reversal activity on P-gp-mediated MDR. Only to find, ES-18 has a strong interaction with P-gp protein than VRP, in consistent with the sequence from experiments.Overall, the present study demonstrated that ES-18 exhibited potential in reversing P-gp mediated MDR. Further investigations on the mechanism of action of ES-18 in vivo will be undertaken in the future.
|胡蕊. 对叶大戟二萜类成分逆转肿瘤多药耐药的研究[D]. 北京. 中国科学院大学,2018.|
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