【作者】 潘凤；

【导师】 梁后杰；

【作者基本信息】 第三军医大学 ， 肿瘤学， 2008， 博士

【摘要】 结肠癌是发病率和死亡率较高的恶性肿瘤,结肠癌的耐药现象是影响其化疗疗效和预后的主要原因之一。为克服肿瘤耐药,多年来人们对肿瘤的耐药机制进行了大量的研究。已证实多种肿瘤组织均有NF-κB的高表达,而多种抗癌药物均可激活NF-κB,NF-κB高表达的肿瘤细胞表现为对化疗、射线等介导凋亡的抵抗作用。我们的前期研究发现肿瘤细胞存在着群集耐药现象,结肠癌细胞三维立体培养时NF-κB活性明显高于普通单层培养,NF-κB活性与药物敏感性呈显著负相关,抑制NF-κB活性,可显著增加药物的敏感性,说明NF-κB信号转导通路是介导肿瘤群集耐药的重要途径。近年有作者甚至认为,NF-κB活化可能代表一个比多药耐药基因更为重要的耐药机制。合理设计与NF-κB活化抑制剂联合的化疗方案,将有助于降低肿瘤细胞的抗凋亡阈值,成为逆转肿瘤耐药和化疗增敏新的策略和有效方法。目前,针对抑制NF-κB活性的策略,直接将突变型IκBα基因转移到肿瘤患者体内还存在着许多技术难题尚未克服;反义寡核苷酸治疗有可能导致机体一些重要基因功能的丧失;正常情况下,由于蛋白酶体和钙调节蛋白酶等是细胞周期和细胞功能的重要调节剂,所以其抑制剂的临床应用还存在很大问题;而圈套寡核苷酸无论从技术和经济上目前也很难在临床广泛应用。因此,如何选择高效、安全的NF-κB抑制剂,仍是目前临床医学中有待解决的问题。进一步发掘靶点特异、高效、低毒的药物,寻找天然NF-κB活性抑制剂,将为拮抗NF-κB功能开辟更加广阔的前景。中医中药是我国独有的优势,从中药中提取具有化疗增敏和逆转耐药作用的中药单体,可大大提高肿瘤的治疗效果,具有广阔的应用前景和巨大的经济和社会效益。目前已有许多中药或中药方剂用于肿瘤的治疗,也发现很多中药具有逆转耐药和化疗增敏的作用。有研究发现,青蒿、雷公藤、银黄、黄芪等中药具有抑制NF-κB活化的作用,青蒿素、大黄素、黄芩甙等中药有效成分也有拮抗NF-κB活化的作用。因此,如果能从中药中分离制备与NF-κB及其亚单位作用的有效单体,将为寻找有效的NF-κB抑制剂提供一条新的途径。由于中药组成成分复杂,如能采用有效手段,确定与NF-κB作用的物质基础,分离制备出抑制NF-κB活化的有效单体,将对NF-κB抑制剂的开发具有重要意义。光学生物传感器技术是利用光学原理,在生物传感器的样品池表面包被固定受体,当受体与其配结合体时,以共振角度进入光线衰竭区的激光就会发生共振角度的改变,这一变化通过计算机处理后就可以检测到传感器表面受体与其配体分子之间的相互作用及亲和力。通过特异性的洗脱及回收,即可得到纯度极高的配体分子。本实验在既往研究工作的基础上,以青蒿为研究对象,利用生物传感器技术,制备出青蒿中与NF-κB特异性结合的活性物质,继而对该物质的理化性质及抑制NF-κB活化的能力进行测定;在体外采用肿瘤细胞三维立体培养模型,观察其抑制NF-κB活性及其对凋亡相关蛋白、细胞凋亡和对药物敏感性的影响;同时进一步观察其对动物肿瘤模型化疗增敏和逆转耐药的效果,以期为肿瘤的化疗增敏和逆转耐药提供新的策略,为其他NF-κB活化相关疾病如感染、炎症、移植等的治疗提供新的思路。方法1.应用生物传感器特异性洗脱回收、有机溶剂萃取、硅胶柱色谱、薄层层析等方法对青蒿中NF-κB的特异性抑制剂进行筛选分离提取;2.检测所得物质的理化性质,进行红外光谱、高效液相、核磁共振波谱、质谱等检测,对所得物质进行结构鉴定;3.体外采用结肠癌HT-29细胞三维立体培养模型,观察所得物质对结肠癌细胞球NF-κB活性(EMSA方法)及化疗药物敏感性的影响;4.应用流式细胞技术检测所得物质对结肠癌细胞球细胞周期、细胞凋亡及凋亡相关蛋白Bcl-2、FAS、FAS-L的影响;5.体内采用结肠癌HT-29细胞裸鼠皮下移植瘤模型,测量裸鼠移植瘤的肿瘤体积及称取肿瘤重量,计算瘤体积和瘤重抑制率,观察所得物质与化疗药物联合应用后对肿瘤生长影响;6.应用EMSA、免疫组织化学的方法检测不同处理组肿瘤组织中NF-κB活性及凋亡相关蛋白的变化情况。结果1.成功分离提取制备得到青蒿中能与NF-κB特异性结合的有效活性物质,并测定其理化性质为淡黄色粉针状,略溶于水,溶于乙醇,易溶于氯仿、乙酸乙酯,熔点在202-203℃。2.红外光谱检测结果,提取得到的化合物IRvKBrmax cm-1在: 3334.7、1701.1、1566.1、1290.3附近有明显振幅波峰。核磁共振波谱结果: 1H-NMR(CDCl3)δppm:3.959(3H,s, OCH3),6.207(1H,s,OH),6.276(1H,d,J=9.2,H-3),6.852(1H,s,H-8),6.923(1H,s,H-5),7.608(1H,d,J=10,H-4)。13C-NMR(CDCl3)δppm:161.383 (C-2),113.433(C-3),143.236(C-4),107.542(C-5),149.733(C-6) , 150.301(C-7) , 103.213(C-8) , 144.029(C-9) , 111.509(C-10) ,56.434(-OCH3)。分析红外光谱、核磁共振波谱及质谱等检测结果,初步得出所得物质的结构并将其暂时命名为AH-I。3. AH-I应用后,结肠癌HT-29细胞球NF-κB的活性受到抑制,AH-I与5-FU联合应用对细胞球NF-κB活性的抑制作用更强。4.单独AH-I应用后,与对照组比较,结肠癌HT-29细胞球细胞周期的变化不明显,大部分细胞位于G1期;而A H-I与5-FU联合应用后,结肠癌HT-29细胞球细胞周期明显受到影响,G1期细胞明显减少,细胞向S期转化,且AH-I应用剂量越大效应越明显。5. AH-I与5-FU联合应用后,结肠癌HT-29细胞球细胞凋亡率与单独化疗组比较明显增加,且随着AH-I应用剂量的增加,细胞球细胞凋亡率增加越明显,存在剂量依赖效应。6. AH-I与5-FU联合应用后,结肠癌HT-29细胞球抗凋亡蛋白Bcl-2的表达与单独化疗组比较有所减少,而促凋亡蛋白FAS、FAS-L的表达较单独化疗组明显增多,且都具有剂量依赖效应。7.结肠癌HT-29细胞球应用AH-I后,对化疗药物5-FU的敏感性增强,且随着AH-I应用剂量的增加,结肠癌细胞球对化疗药物的敏感性增加越明显,具有剂量依赖效应。8.体内裸鼠移植瘤模型,AH-I与5-FU联合应用后肿瘤的瘤体积与单独化疗组比较有所减小,肿瘤的瘤重与单独化疗组比较也有所减轻,瘤体积抑制率、瘤重抑制率与单独5-FU组比较均明显增加,肿瘤生长受到抑制。9.体内裸鼠移植瘤模型应用AH-I后,肿瘤组织NF-κB活性受到抑制,且AH-I与5-FU联合应用组肿瘤组织的NF-κB活性的抑制较单独化疗组更明显,且同样具有剂量依赖效应。10. AH-I与5-FU联合应用后,裸鼠移植瘤肿瘤组织中抗凋亡蛋白Bcl-2表达减少,促凋亡蛋白FAS、FAS-L的表达增加,且与AH-I的应用剂量有关,AH-I应用剂量越大,抗凋亡蛋白Bcl-2表达减少及促凋亡蛋白FAS、FAS-L的表达增加更明显,都具有剂量依赖效应。结论1.成功分离得到青蒿中NF-κB的特异性抑制剂,分析其结构后暂时命名为AH-I。2. AH-I对结肠癌HT-29细胞球的NF-κB活性有抑制,可增加结肠癌细胞球对化疗药物5-FU的敏感性。3. AH-I与5-FU联合应用后,对结肠癌细胞球细胞周期、凋亡相关蛋白的表达产生影响,促进细胞凋亡的发生。4. AH-I与5-FU联合应用后,在体结肠癌裸鼠移植瘤的肿瘤生长减慢,增强5-FU化疗的疗效。5. AH-I与5-FU联合应用于结肠癌裸鼠移植瘤,可抑制肿瘤组织NF-κB活性,降低抗凋亡蛋白Bcl-2的表达,增强促凋亡蛋白FAS、FAS-L的表达。更多还原

【Abstract】 Background and ObjectiveAnticancer drug resistance is the underlying cause of treatment failure in a significant number of patients with cancer. The transcription factor NF-kB is reportedly activated by anti-cancer chemotherapeutic compounds in many cancer cell lines and NF-kB activation is one mechanism by which tumors become resistant to apoptosis. In addition to this constitutive activity, the accumulating evidences has been clearly demonstrated that many anti-cancer agents can activate NF-κB in cancer cells. Either constitutive or treatment-induced activity, NF-κB mainly both acts as an inhibitor of apoptosis. Indeed, inhibition of NF-κB by genetic or chemical inhibitors can induces the apoptosis of various tumor cells and/ or restores the apoptotic response after treatment with ionizing radiations or chemotherapeutic agents, thus reversing NF-κB -linked radiotherapy or chemotherapy resistance in many models. Therefore, a precise knowledge of the signalling pathways controlling NF-κB as well as of the NF-κB target genes is essential in order to define precisely the field of application of anti- NF-κB drugs for the treatment of human cancers.Activation of NF-κB in cancer cells has been shown to attenuate apoptosis induced by chemotherapy or radiotherapy. The results of our previous studies suggest that NF-κB is an important molecular target for enhancing chemosensitivity. Consequently, the development of novel agents blocking the NF-κB activity has become a major goal for numerous laboratories and companies.Preclinical studies are being conducted regarding the inhibition of NF-κB activation in several laboratories.Tumor spheroids represent a excellent model to study drug resistance and in particular the so-called multicellular-mediated resistance(MCR) to anticancer agents.Classically,this type of resistance has been demonstrated in EMT6 tumors in mice in which resistance was inherently induced, whereas it was completely lost when the cancer cells were isolated and grown in monolayers; multicellular resistance, however, could be fully recapitulated when cells were cultured as multicellular spheroids.Chinese medicine is our unique advantage, there are many traditional Chinese drugs or treatments being treated to tumor.It has been found that many traditional Chinese drugs can augmented sensitivity to chemotherapeutic drugs and decrease cancer cells resistance to chemotherapy. Many of studies found that abrotani herba, triptolide, astragalus and other traditional Chinese drugs can inhibit the activation of NF-κB, artemisinin, rhein, baicalin, and other active ingredients of traditional Chinese drugs also can inhibit NF-κB activation. Therefore, it will provide a new way to find effective inhibitor of NF-κB if we could extract the inhibitor of NF-κB from the traditional Chinese drugs. Because of the complex composition of traditional Chinese drug, it is great significance to chose a effective method to obtain the inhibitor of NF-κB extracting from traditional Chinese drugs. So in this study,we used of biosensor technology, extracted the inhibitor of NF-κB from abrotani herba and identificated its structure .Secondly, studed the effection of the inhibitor on NF-κB activity , sensitivity to 5-FU,apoptosis and expression of apoptostic proteins in HT-29 spheroids.Finally,we studied effetion of the inhibitor in chemotherapy in xenograft experiment.Methods1. The specific inhibitor of NF-κB from abrotani herba was extracted by biosensor technology, organic solvent extraction, silica gel column chromatography, thin layer chromatography and other methods;2. The physico-chemical property of the specific inhibitor was tested .Then infrared Spectrometer(IR), high-pressure liquid chromatograph(HPLC), nuclear magnetic resonance spectroscopy(NMR), mass spectrometry(MS) were tested, and the structure was identificated;3. In vitro,effection of the inhibitor on NF-κB activity and the impact of chemotherapy sensitivity were studied in three-dimensiona colon cancer HT-29 cells;4. The cell cycle distribution,apoptosis and expression of apoptostic protein Bcl-2, FAS, and FAS-L on three-dimensiona HT-29 cells were detected by administration the NF-κB inhibitor extracting from abrotani herba with flow cytometry;5. In vivo, useing of HT-29 cells transplantation tumor subcutaneously in nude mice model, the effect of the inhibitor combination with chemotherapy in tumor growth was studied;6. With EMSA and immunohistochemical method, activity of NF-κB and expression of apoptostic protein Bcl-2, FAS, and FAS-L on tumors in xenograft experiments combination inhibitor with 5-FU were detected.Results1. The specific inhibitor of NF-κB from abrotani herba was successfully extracted ,and the physico-chemical property of the specific inhibitor was that slightly soluble in water, soluble in ethanol, soluble in chloroform, soluble in ethyl acetate, in the melting point 202-203℃.2. IR, NMR and MS of the inhibitor of NF-κB extracting from abrotani herba were tested, the structure of the inhibitor was identified,and it named AH-I temporary.Results followed: IRvKBrmax cm-1: 3334.7,1701.1,1566.1,1290.3. 1H-NMR(CDCl3)δppm:3.959(3H,s,OCH3),6.207(1H,s,OH),6.276(1H,d,J=9.2,H-3),6.852(1H,s,H-8),6.923(1H,s,H-5),7.608(1H,d,J=10,H-4). 13C-NMR(CDCl3)δppm:161.383(C-2),113.433(C-3),143.236(C-4),107.542(C-5),149.733(C-6),150.301(C-7),103.213(C-8),144.029(C-9),111.509(C-10),56.434(-OCH3).3. Administration AH-I, NF-κB activity in three-dimensiona HT-29 cells was inhibited, and combination AH-I with 5 - FU, the activity of NF-κB inhibition was stronger than 5– FU alone.4.Compared with the control, cells majority in the G1 phase and cell cycle was not changed significantly in three-dimensiona HT-29 cells treated with AH-I alone. While AH-I and 5 - FU combination, cell cycle obviously were changed in three-dimensiona HT-29 cells, the number of cells in G1 phase was decreased significantly ,and the number of cells in S phase was increased, both with the manner of dose-independent.5. AH-I and 5 - FU combination, apoptosis in three-dimensiona HT-29 cells was significantly increased compared with chemotherapy alone, and as the AH-I dose increasing, rates of apoptosis in three-dimensiona HT-29 cells were more obvious increased , with a dose-dependent effect.6. Combination AH-I plus 5 - FU, expression of anti-apoptosis protein Bcl-2 was decreased compared with chemotherapy alone group. But expression of pro-apoptotic protein FAS and FAS-L were increased significantly than chemotherapy alone group and both in a dose-dependent manner.7. Administration of AH-I on three-dimensiona HT-29 cells, the sensitivity to 5 - FU was enhanced, and with the AH-I dose increasing, the sensitivity to 5 - FU of three-dimensiona HT-29 cells was more obvious , and also in a dose-dependent manner8. In nude mice, AH-I and 5 - FU combination, volumes of tumors were decreased than 5-FU alone,weights of tumours were in the same changes with volumes ,growth of tumour was inhibited.9. In nude mice , NF-κB activity in tumour was inhibited in AH-I alone group.Combination AH-I and 5 - FU , NF-κB activity in tumour was inhibited more obviously than chemotherapy alone, also with a dose-dependent effect.10. In nude mice , combination AH-I and 5 - FU ,expression of the anti-apoptotic protein Bcl-2 in tumour was decreased ,but expression of apoptotic protein FAS and FAS-L in tumour were increased , and all of them were in dose-dependent effect.Conclusion1. The specific inhibitor of NF-κB from abrotani herba was successfully extracted ,then the inhibitor was identified and was named of AH-I temporary.2. NF-κB ativity was inhibited and sensitivity to 5 - Fu was enhanced in HT-29 spheroids by AH-I. 3. AH-I combination with 5–FU , cell cycle distribution was effected and expression of apoptostic proteins Bcl-2, FAS and FAS-L were effected aslo, apoptosis was induced in HT-29 spheroids in vitro.4. AH-I combination with 5–FU , growth of tumours was inhibited and effect of chemotherapy was enhanced in xenograft experiment.5. AH-I combination with 5–FU , cell cycle distribution was effected and expression of apoptostic proteins Bcl-2, FAS and FAS-L were effected aslo, apoptosis was induced in xenograft experiment in vivo.更多还原