二种雷公藤属药用植物中逆转肿瘤多药耐药性的萜类成分发掘和构效关系研究

Mutidrug resistance (MDR) was a common phenomena and difficulty in the chemotherapy of tumor. The efflux of anti-tumor drugs meditated by P-glycoprotein (P-gp) was the main problem of MDR.The inhibition of P-gp was acknowledged as an efficient strategy for the MDR reversal. However, no specific drugs of P-gp inhibitors have been exploited by far..The diterpenes and sesquiterpenes from the plants of Tripterygium genus have been proved to possess significant activities of anti-tumor, MDR reversal, and P-gp inhibition. T. regelii and T. Hypoglaucum were traditional Chinese medicine and poisonous plants of Tripterygium genus. In preliminary study, we found that the 2-epitripdiolide (diterpene) identified from T. Hypoglaucum exhibited 300 folds more potent cytotoxity against MDR KBvin cells than paclitaxel (positive control).To continue the study, the aim of project was to isolate and identify constituents of novel diterpenes and sesquiterpenes from T. regelii and T. Hypoglaucum with various of modern chromatography and NMR technologies and under the guidance of LC/MS/MS. Meanwhile, 2-epitripdiolide was planned to be structurally modified. All the natural and synthesized terpenes were evaluated activities of anti-tumor and MDR reversal using MCF-7, MCF-7/Adr (adriamycin-resistant and P-gp overexpression strain), IL-60, and IL-60/Adr(adriamycin-resistant and P-gp overexpression strain) cells. The compounds which were proved to reverse MDR of MCF-7/Adr and IL-60/Adr were further tested its effect on expression and transporting function of P-gp of MCF-7/Adr and IL-60/Adr. And the binding of compounds inhibiting P-gp expression or function with P-gp were finally validated by surface plasmon resonance (SPR) technology in vitro. it was expected that the the difference of chemical constituents between T. regelii and T. Hypoglaucum would be revealed. And the most concerned aspect was that the chemical compounds with MDR reversal activities and targeting at P-gp can be identified from the two species of medical plants. As a by-product, we also hoped that anti-tumor compounds can be founded in the project. Based on the data of the activity screening, the possible MDR reversal mechanism of lead compounds and structure activity relationship would be discussed and established.

多药耐药性是肿瘤化疗中的常见现象和难题。P-糖蛋白介导的药物转运外排是大多数肿瘤耐药的主要原因，抑制P-糖蛋白是公认有效的逆转肿瘤耐药策略之一。目前仍无特效的P-糖蛋白抑制剂类药物。.雷公藤属植物中的二萜和倍半萜具有显著逆转（抗）耐药性（肿瘤）和抑制P-糖蛋白活性。传统中药昆明山海棠和东北雷公藤是该属有毒植物。前期研究中，从昆明山海棠中分离鉴定的二萜2-表雷公藤乙素，对KBvin耐药株的细胞毒活性是紫杉醇的约300倍。本项目拟在LC/MS指导分离下，利用各种色谱层析和NMR技术，分离和鉴定二种药材中结构新颖的二萜和倍半萜类成分；并对2-表雷公藤乙素进行结构修饰。得到的萜类进行逆转（抗）耐药性（肿瘤）活性评价，分析其对耐药株 P-糖蛋白表达和转运活性的影响，并用SPR技术验证。揭示二种药材化学成分的差异；探讨以P-糖蛋白为靶点，逆转（抗）耐药性（肿瘤）的活性萜类成分及可能的作用机理和构效关系

多药耐药性是肿瘤化疗中的常见现象和难题。P-糖蛋白介导的药物转运外排是大多数肿瘤耐药的主要原因，抑制P-糖蛋白是公认有效的逆转肿瘤耐药策略之一。本项目根据前期和文献调研，以两种雷公藤属植物（雷公藤和昆明山海棠）和大戟属植物千金子为研究对象，从上述三种植物中共分离鉴定了170个化合物，其中新化合物37个，并以雷公藤甲素为母体修饰得到了10个衍生物。对分离得到的二萜，倍半萜及结构修饰得到的雷公藤甲素衍生物进行了抗HepG/Adr和HepG细胞增殖活性评价，雷公藤甲素具有显著抗HepG2细胞增殖活性 (IC50=0.203μM)，修饰得到的雷公藤甲素衍生物1-10也具有显著的抗HepG2细胞增殖活性（IC50值范围: 0.08-6.5μM)。雷公藤甲素及修饰得到的类似物2, 5, 8, 9和10对HepG2/Adr细胞株也有显著的细胞毒活性，IC50分别是1.8, 6.5, 8.6, 3.2, 1.7和4.2 μM。对分离得到的倍半萜和二萜进行了逆转HepG/Adr多药耐药活性评价，发现了发现了4个具有显著逆转HepG2/Adr细胞MDR活性的二氢沉香呋喃型倍半萜和3个千金烷型二萜，对代表性活性化合物chiapen D（分离自昆明山海棠）和euphorantin I（分离自千金子）的逆转肿瘤MDR活性作用机理进行了研究，发现chiapen D和euphorantin I均通过与抗肿瘤药物阿霉素（Adr）竞争性结合P-糖蛋白逆转肿瘤MDR。同时结合雷公藤属植物的以抗炎为主的传统用药特点，对雷公藤和昆明山海棠中分离得到的二萜进行了抗炎活性评价，发现了4个具有显著抗炎活性的松香烷型二萜，并对代表性活性化合物tritophenolide和quinone 21的抗炎活性机理进行了探索，发现与其抑制p38、JNK、ERK和IκBα磷酸化水平有关。C环的对醌结构可能是松香烷型二萜抗炎的关键活性基团，二氢沉香呋喃型倍半萜和千金烷型二萜逆转MDR的构效关系尚不明确。