本文選題：雷公藤內(nèi)酯醇 + CDK7　； 參考：《中國(guó)科學(xué)院上海藥物研究所》2016年博士論文

【摘要】：腫瘤耐藥是腫瘤治療失敗的重要原因。在臨床中幾乎所有的化療藥物都不可避免地產(chǎn)生腫瘤耐藥,導(dǎo)致治療失敗,嚴(yán)重影響腫瘤患者的生存質(zhì)量和生存時(shí)間。無(wú)數(shù)科研工作者致力于探究藥物敏感性機(jī)制及腫瘤耐藥機(jī)制,以期開發(fā)出有效的治療策略和新型抗腫瘤藥物,從而克服這一巨大阻礙。目前,針對(duì)不同類型的耐藥患者,已有抗腫瘤藥物或者藥物聯(lián)合應(yīng)用正處于臨床試驗(yàn)中。本課題系統(tǒng)研究了雷公藤內(nèi)酯醇和雙重靶向微管-拓?fù)洚悩?gòu)酶II(topoisomerase II,Top2)抑制劑YCH337兩個(gè)小分子化合物的抗腫瘤作用、抗耐藥作用及其作用機(jī)制,為新型抗腫瘤藥物及抗耐藥藥物研發(fā)提供了新的方向。本課題在我們已有研究基礎(chǔ)上進(jìn)一步探究了雷公藤內(nèi)酯醇的抗多藥耐藥作用并深入解析其抗腫瘤及抗耐藥作用機(jī)制。已有的研究表明雷公藤內(nèi)酯醇能夠通過(guò)激活CDK7引起Rpb1發(fā)生降解。我們的研究結(jié)果顯示,雷公藤內(nèi)酯醇能夠有效殺傷多藥耐藥細(xì)胞KB/VCR、MES-SA/DX5以及K562/A02,平均耐藥指數(shù)(Resistance Factor,RF)為0.77。對(duì)KB/VCR和MES-SA/DX5的生長(zhǎng)增殖抑制作用比親本細(xì)胞強(qiáng)近2倍,RF分別為0.55和0.54。藥物轉(zhuǎn)運(yùn)蛋白P-糖蛋白(P-glycoprotein,P-gp)過(guò)表達(dá)是腫瘤產(chǎn)生多藥耐藥的一個(gè)重要原因,但是雷公藤內(nèi)酯醇不是通過(guò)影響P-gp發(fā)揮抗多藥耐藥作用的。雷公藤內(nèi)酯醇1μM處理KB/VCR 90 min不影響P-gp藥物轉(zhuǎn)運(yùn)功能;雷公藤內(nèi)酯醇50 nM處理長(zhǎng)時(shí)間(≥36 h)才會(huì)引起P-gp蛋白水平和MDR1 mRNA水平下調(diào),而Rpb1在4 h就開始發(fā)生降解,且通過(guò)CDK7抑制劑逆轉(zhuǎn)Rpb1降解能夠逆轉(zhuǎn)P-gp水平。因此,雷公藤內(nèi)酯醇對(duì)P-gp的影響是其轉(zhuǎn)錄抑制作用的后續(xù)效應(yīng)。腫瘤干性是腫瘤耐藥的另一個(gè)重要因素,KB/VCR與KB相比克隆形成能力增加~10%,雷公藤內(nèi)酯醇能夠有效抑制KB及KB/VCR的克隆形成能力,IC50分別是0.68 nM和0.41nM。同時(shí),能下調(diào)腫瘤干性相關(guān)轉(zhuǎn)錄因子c-Myc及c-myc mRNA的水平。但是雷公藤內(nèi)酯醇對(duì)c-myc的下調(diào)可能也不是其發(fā)揮抗多藥耐藥作用的主要原因,因?yàn)檫^(guò)表達(dá)c-myc并不會(huì)影響雷公藤內(nèi)酯醇對(duì)kb和kb/vcr的增殖生長(zhǎng)抑制作用。雷公藤內(nèi)酯醇在親本細(xì)胞和多藥耐藥細(xì)胞中發(fā)揮抗腫瘤作用及抗耐藥作用的機(jī)制是一致的,均通過(guò)上調(diào)第170位蘇氨酸磷酸化(p-t170-cdk7)水平使cdk7激活,引起rpb1降解。用cdk7選擇性抑制劑bs-181預(yù)處理,能夠逆轉(zhuǎn)或延緩雷公藤內(nèi)酯醇引起的rpb1降解,并部分逆轉(zhuǎn)雷公藤內(nèi)酯醇的增殖生長(zhǎng)抑制作用。采用質(zhì)譜分析我們發(fā)現(xiàn)雷公藤內(nèi)酯醇引起rpb1的ctd末端第5位絲氨酸發(fā)生磷酸化的一個(gè)具體位點(diǎn)為1878位絲氨酸。同時(shí),我們發(fā)現(xiàn)xpb以及p44的缺失不影響雷公藤內(nèi)酯醇引起的rpb1降解及細(xì)胞增殖生長(zhǎng)抑制作用,從而否定了此前關(guān)于xpb是雷公藤內(nèi)酯醇抗腫瘤作用靶點(diǎn)的結(jié)論。目前,雷公藤內(nèi)酯醇的衍生物minnelide對(duì)胰腺癌和肝癌的臨床研究正在進(jìn)行當(dāng)中。我們的研究結(jié)果為擴(kuò)大雷公藤內(nèi)酯醇及其衍生物潛在的臨床應(yīng)用范圍提供了支持,為進(jìn)一步探究并最終闡明雷公藤內(nèi)酯醇產(chǎn)生抗腫瘤作用的靶點(diǎn)提供了新的線索。本課題另一方面的工作是對(duì)具有抗耐藥作用的全新化合物ych337的抗腫瘤作用和機(jī)制的研究。我們與藥物化學(xué)楊春皓研究員研究組合作發(fā)現(xiàn)α-咔啉新衍生物ych337具有很好的抗腫瘤作用。我們發(fā)現(xiàn)ych337能夠同時(shí)靶向微管和top2,為微管-top2雙重抑制劑。微管和top2都是重要的抗腫瘤靶點(diǎn),微管抑制劑與top2抑制劑是臨床上廣泛應(yīng)用的抗腫瘤藥物,常常聯(lián)合應(yīng)用;目前沒(méi)有同時(shí)靶向微管與top2的藥物進(jìn)入臨床研究�？鼓[瘤作用研究顯示,ych337對(duì)19株不同組織來(lái)源的不同類型的人腫瘤細(xì)胞均具有良好的增殖生長(zhǎng)抑制作用,平均ic50為0.3μm。ych337能夠顯著抑制裸鼠ht-29移植瘤的生長(zhǎng)。此外,ych337對(duì)不同耐藥機(jī)制的腫瘤耐藥細(xì)胞以及親本細(xì)胞具有同等程度的增殖生長(zhǎng)抑制作用。機(jī)制研究顯示,ych337通過(guò)結(jié)合到秋水仙堿位點(diǎn),抑制微管聚合,使腫瘤細(xì)胞產(chǎn)生多極紡錘體,引起明顯的m期阻滯;同時(shí),ych337還抑制top2的活性,引起dna雙鏈斷裂,導(dǎo)致γh2ax蓄積增加。ych337對(duì)微管的抑制作用強(qiáng)于其對(duì)top2活性的抑制作用。用0.1μmych337處理hela細(xì)胞1h,或者1μmych337處理hela細(xì)胞15min就能引起明顯的微管解聚,但是較高藥物濃度(0.2μm)和較長(zhǎng)的處理時(shí)間(30min)才能發(fā)揮top2抑制作用。ych337在0.2μm時(shí)誘導(dǎo)可逆的有絲分裂阻滯,而dna損傷作用不可逆;在高濃度(≥0.5μM)時(shí),有絲分裂阻滯和DNA損傷均不可逆。YCH337通過(guò)激活內(nèi)源性及外源性凋亡通路,誘導(dǎo)Caspase 9/8/3剪切激活,導(dǎo)致腫瘤細(xì)胞發(fā)生凋亡。YCH337還降低MCL-1、c IAP1及XIAP等抗凋亡蛋白的水平。YCH337誘導(dǎo)凋亡與傳統(tǒng)微管抑制劑長(zhǎng)春新堿(vincristine;VCR)作用相似,與Top2抑制劑依托泊苷(etoposide;VP-16)不同;并且當(dāng)VP-16與VCR聯(lián)合應(yīng)用時(shí),VP-16減弱了VCR誘導(dǎo)凋亡的能力及下調(diào)抗凋亡蛋白的能力。因此,單純的聯(lián)合應(yīng)用VCR與VP-16無(wú)法達(dá)到好的治療效果,雙重靶向于微管和Top2可能為聯(lián)合用藥面臨的多種問(wèn)題提供新的解決方案。綜上所述,本課題對(duì)兩個(gè)不同的抗腫瘤耐藥化合物雷公藤內(nèi)酯醇及YCH337的作用及機(jī)制進(jìn)行了系統(tǒng)研究,為新型抗腫瘤藥物開發(fā)提供了新的思路。隨著我們對(duì)腫瘤發(fā)生發(fā)展及耐藥的深入了解,合理設(shè)計(jì)并開發(fā)新型抗腫瘤藥物,在取得良好治療效果的同時(shí),也能夠克服腫瘤耐藥給腫瘤治療帶來(lái)的巨大阻礙,為腫瘤患者帶來(lái)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.

【學(xué)位授予單位】：中國(guó)科學(xué)院上海藥物研究所
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R96

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本文編號(hào)：1798369