抗肿瘤耐药化合物雷公藤内酯醇与YCH337的作用及机制研究

发布时间：2018-04-24 21:44

本文选题：雷公藤内酯醇 + CDK7　；参考：《中国科学院上海药物研究所》2016年博士论文

【摘要】：肿瘤耐药是肿瘤治疗失败的重要原因。在临床中几乎所有的化疗药物都不可避免地产生肿瘤耐药,导致治疗失败,严重影响肿瘤患者的生存质量和生存时间。无数科研工作者致力于探究药物敏感性机制及肿瘤耐药机制,以期开发出有效的治疗策略和新型抗肿瘤药物,从而克服这一巨大阻碍。目前,针对不同类型的耐药患者,已有抗肿瘤药物或者药物联合应用正处于临床试验中。本课题系统研究了雷公藤内酯醇和双重靶向微管-拓扑异构酶II(topoisomerase II,Top2)抑制剂YCH337两个小分子化合物的抗肿瘤作用、抗耐药作用及其作用机制,为新型抗肿瘤药物及抗耐药药物研发提供了新的方向。本课题在我们已有研究基础上进一步探究了雷公藤内酯醇的抗多药耐药作用并深入解析其抗肿瘤及抗耐药作用机制。已有的研究表明雷公藤内酯醇能够通过激活CDK7引起Rpb1发生降解。我们的研究结果显示,雷公藤内酯醇能够有效杀伤多药耐药细胞KB/VCR、MES-SA/DX5以及K562/A02,平均耐药指数(Resistance Factor,RF)为0.77。对KB/VCR和MES-SA/DX5的生长增殖抑制作用比亲本细胞强近2倍,RF分别为0.55和0.54。药物转运蛋白P-糖蛋白(P-glycoprotein,P-gp)过表达是肿瘤产生多药耐药的一个重要原因,但是雷公藤内酯醇不是通过影响P-gp发挥抗多药耐药作用的。雷公藤内酯醇1μM处理KB/VCR 90 min不影响P-gp药物转运功能;雷公藤内酯醇50 nM处理长时间(≥36 h)才会引起P-gp蛋白水平和MDR1 mRNA水平下调,而Rpb1在4 h就开始发生降解,且通过CDK7抑制剂逆转Rpb1降解能够逆转P-gp水平。因此,雷公藤内酯醇对P-gp的影响是其转录抑制作用的后续效应。肿瘤干性是肿瘤耐药的另一个重要因素,KB/VCR与KB相比克隆形成能力增加~10%,雷公藤内酯醇能够有效抑制KB及KB/VCR的克隆形成能力,IC50分别是0.68 nM和0.41nM。同时,能下调肿瘤干性相关转录因子c-Myc及c-myc mRNA的水平。但是雷公藤内酯醇对c-myc的下调可能也不是其发挥抗多药耐药作用的主要原因,因为过表达c-myc并不会影响雷公藤内酯醇对kb和kb/vcr的增殖生长抑制作用。雷公藤内酯醇在亲本细胞和多药耐药细胞中发挥抗肿瘤作用及抗耐药作用的机制是一致的,均通过上调第170位苏氨酸磷酸化(p-t170-cdk7)水平使cdk7激活,引起rpb1降解。用cdk7选择性抑制剂bs-181预处理,能够逆转或延缓雷公藤内酯醇引起的rpb1降解,并部分逆转雷公藤内酯醇的增殖生长抑制作用。采用质谱分析我们发现雷公藤内酯醇引起rpb1的ctd末端第5位丝氨酸发生磷酸化的一个具体位点为1878位丝氨酸。同时,我们发现xpb以及p44的缺失不影响雷公藤内酯醇引起的rpb1降解及细胞增殖生长抑制作用,从而否定了此前关于xpb是雷公藤内酯醇抗肿瘤作用靶点的结论。目前,雷公藤内酯醇的衍生物minnelide对胰腺癌和肝癌的临床研究正在进行当中。我们的研究结果为扩大雷公藤内酯醇及其衍生物潜在的临床应用范围提供了支持,为进一步探究并最终阐明雷公藤内酯醇产生抗肿瘤作用的靶点提供了新的线索。本课题另一方面的工作是对具有抗耐药作用的全新化合物ych337的抗肿瘤作用和机制的研究。我们与药物化学杨春皓研究员研究组合作发现α-咔啉新衍生物ych337具有很好的抗肿瘤作用。我们发现ych337能够同时靶向微管和top2,为微管-top2双重抑制剂。微管和top2都是重要的抗肿瘤靶点,微管抑制剂与top2抑制剂是临床上广泛应用的抗肿瘤药物,常常联合应用;目前没有同时靶向微管与top2的药物进入临床研究。抗肿瘤作用研究显示,ych337对19株不同组织来源的不同类型的人肿瘤细胞均具有良好的增殖生长抑制作用,平均ic50为0.3μm。ych337能够显著抑制裸鼠ht-29移植瘤的生长。此外,ych337对不同耐药机制的肿瘤耐药细胞以及亲本细胞具有同等程度的增殖生长抑制作用。机制研究显示,ych337通过结合到秋水仙碱位点,抑制微管聚合,使肿瘤细胞产生多极纺锤体,引起明显的m期阻滞;同时,ych337还抑制top2的活性,引起dna双链断裂,导致γh2ax蓄积增加。ych337对微管的抑制作用强于其对top2活性的抑制作用。用0.1μmych337处理hela细胞1h,或者1μmych337处理hela细胞15min就能引起明显的微管解聚,但是较高药物浓度(0.2μm)和较长的处理时间(30min)才能发挥top2抑制作用。ych337在0.2μm时诱导可逆的有丝分裂阻滞,而dna损伤作用不可逆;在高浓度(≥0.5μM)时,有丝分裂阻滞和DNA损伤均不可逆。YCH337通过激活内源性及外源性凋亡通路,诱导Caspase 9/8/3剪切激活,导致肿瘤细胞发生凋亡。YCH337还降低MCL-1、c IAP1及XIAP等抗凋亡蛋白的水平。YCH337诱导凋亡与传统微管抑制剂长春新碱(vincristine;VCR)作用相似,与Top2抑制剂依托泊苷(etoposide;VP-16)不同;并且当VP-16与VCR联合应用时,VP-16减弱了VCR诱导凋亡的能力及下调抗凋亡蛋白的能力。因此,单纯的联合应用VCR与VP-16无法达到好的治疗效果,双重靶向于微管和Top2可能为联合用药面临的多种问题提供新的解决方案。综上所述,本课题对两个不同的抗肿瘤耐药化合物雷公藤内酯醇及YCH337的作用及机制进行了系统研究,为新型抗肿瘤药物开发提供了新的思路。随着我们对肿瘤发生发展及耐药的深入了解,合理设计并开发新型抗肿瘤药物,在取得良好治疗效果的同时,也能够克服肿瘤耐药给肿瘤治疗带来的巨大阻碍,为肿瘤患者带来新的希望。
[Abstract]:Tumor resistance is an important reason for the failure of cancer treatment. In clinical, almost all chemotherapeutic drugs inevitably produce drug resistance, resulting in the failure of the treatment, which seriously affects the quality of life and the survival time of the cancer patients. Numerous researchers have been devoted to exploring the mechanism of drug sensitivity and the mechanism of tumor resistance in order to develop effective drugs. At present, the combination of antitumor drugs or drugs has been in clinical trials for different types of drug resistant patients. The subject systematically studied the Lei Gongteng lactone and the dual target microtubule II (topoisomerase II, Top2) inhibitor YCH3 37 the anti-tumor effect, anti drug resistance and its mechanism of action of two small molecular compounds provide a new direction for the development of new antitumor drugs and anti drug resistant drugs. On the basis of our research, we further explored the anti multidrug resistance of Lei Gongteng lactone alcohol and deeply analyze the mechanism of its anti-tumor and anti drug resistance. Previous studies have shown that triptolide can degrade Rpb1 by activating CDK7. Our results show that triptolide can effectively kill multidrug resistant cells KB/VCR, MES-SA/DX5 and K562/A02, and the average resistance index (Resistance Factor, RF) is the inhibitory ratio of 0.77. to KB/VCR and MES-SA/DX5. The parent cells are nearly 2 times more than 2 times, and the overexpression of RF 0.55 and 0.54. drug transporter P- glycoprotein (P-glycoprotein, P-gp) is an important cause of multidrug resistance in cancer. But triptolide does not exert anti multidrug resistance by affecting P-gp. The treatment of KB/VCR 90 min by 1 u M of triptolide does not affect the drug transport of P-gp. Function: the treatment of Lei Gongteng lactone alcohol 50 nM for a long time (> 36 h) can cause the level of P-gp protein and MDR1 mRNA down, and Rpb1 begins to degrade in 4 h, and the reverse Rpb1 degradation through CDK7 inhibitor can reverse P-gp level. Therefore, the effect of Lei Gongteng lactone on P-gp is the follow-up effect of its transcriptional inhibition. Another important factor in tumor resistance is that KB/VCR increases the clone formation ability of ~10% compared with KB. Triptolide can effectively inhibit the clonogenic ability of KB and KB/VCR. IC50 is 0.68 nM and 0.41nM., respectively, and can downregulate the level of c-Myc and c-myc mRNA of the tumor stem related transcription factors. But triptolide downregulation the c-myc. It may not be the main cause of its anti multidrug resistance, because overexpression of c-myc does not affect the inhibitory effect of Lei Gongteng lactone on the proliferation and growth of KB and kb/vcr. The mechanism of the antitumor and anti drug resistance of Lei Gongteng lactone in parental and multidrug resistant cells is consistent, all through up up 170th bits The level of threonine phosphorylation (p-t170-cdk7) activates Cdk7 and causes rpb1 degradation. The degradation of rpb1 caused by Triptolide can be reversed or delayed by bs-181 pretreatment with Cdk7 selective inhibitor, and the inhibitory effect of triptolide on proliferation and growth of triptolide is partly reversed. We found that triptolide induced the CTD terminal of rpb1 in rpb1. A specific site for the phosphorylation of fifth serine was 1878 serine. At the same time, we found that the deletion of XPB and p44 did not affect rpb1 degradation and cell proliferation inhibition induced by triptolide, which denies the previous conclusion that XPB is the target of the anti swelling tumor of triptolide. The clinical study of pancreatic and liver cancer derivatives, minnelide, is in progress. Our results provide support for the potential clinical applications of triptolide and its derivatives, and provide new clues for further exploring and ultimately clarifying the target of triptolide to produce antitumor targets. One aspect of the work is to study the anti-tumor effect and mechanism of a new compound, ych337, which is resistant to resistance. We combined with researcher Yang Chunhao to find that the new derivative of alpha carbazoline, ych337, has a good antitumor effect. We found that ych337 can target microtubules and top2 at the same time for the double inhibition of microtubule -top2. Microtubules and top2 are all important anti-tumor targets. Microtubule inhibitors and top2 inhibitors are widely used in clinical antitumor drugs, often combined. There is no drug targeting microtubules and top2 at the same time into clinical study. The antitumor effect study shows that ych337 is fine for different types of human tumors from 19 different tissue sources. The cell had a good inhibitory effect on proliferation and growth. The average IC50 of 0.3 mu m.ych337 could significantly inhibit the growth of HT-29 xenografts in nude mice. In addition, ych337 had the same proliferation inhibition effect on the tumor resistant and parental cells of different drug resistance mechanisms. The mechanism study showed that ych337 was combined with colchicine site, Inhibition of microtubule polymerization causes the tumor cells to produce a multipolar spindle and cause an obvious M phase block; at the same time, ych337 also inhibits the activity of top2 and causes DNA double strand breaks, causing the accumulation of.Ych337 to increase the inhibition of microtubules stronger than the inhibition of top2 activity. 0.1 micron mych337 is used to treat HeLa cell 1H, or 1 mu mych337 to treat HeLa cells. 15min can cause obvious microtubule depolymerization, but higher drug concentration (0.2 mu m) and longer treatment time (30min) can induce the reversible mitotic block induced by.Ych337 at 0.2 u m, while DNA damage is irreversible. At high concentration (> 0.5 u M), the mitotic block and DNA damage are all irreversible.YCH337 through activation. Endogenous and exogenous apoptotic pathways induce Caspase 9/8/3 shear activation, resulting in the apoptosis of tumor cells and the decrease of MCL-1, C IAP1 and XIAP and other anti apoptotic proteins, which are similar to the traditional microtubule inhibitor vincristine (vincristine; VCR), and are different from that of Top2 inhibitor etoposide (etoposide;). When combined with VP-16 and VCR, VP-16 reduces the ability of VCR to induce apoptosis and the ability to reduce anti apoptotic protein. Therefore, the simple combination of VCR and VP-16 can not achieve good therapeutic effect. Dual targeting to microtubule and Top2 may provide new solutions for a variety of problems faced by combined drugs. To sum up, the subject is two The effect and mechanism of triptolide and YCH337, a different antitumor drug resistant compound, have been systematically studied to provide new ideas for the development of new antitumor drugs. With our thorough understanding of the development and drug resistance of tumors, the rational design and development of new antitumor drugs can also be achieved as well as good therapeutic effects. It can overcome the huge obstacles brought by tumor resistance to tumor therapy and bring new hope to cancer patients.

【学位授予单位】：中国科学院上海药物研究所
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96

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