【作者】 薛霞；

【导师】 曲显俊； 娄红祥；

【作者基本信息】 山东大学 ， 药理学， 2011， 博士

【摘要】 研究背景拓扑异构酶是一种真核生物生存所必需的核酶,可介导DNA单链或双链的瞬时断裂和再连接,在DNA复制、转录、重组及染色体分离等多个环节发挥重要作用。根据其诱导DNA断裂方式的不同,分为拓扑异构酶Ⅰ和拓扑异构酶Ⅱ,以拓扑异构酶为靶点的抗癌作用成为化疗药物研究的一个重要方向。临床上许多抗肿瘤药物以拓扑异构酶为作用靶点,取得了一定临床疗效。但在应用过程中仍出现一些问题：多药耐药性的产生,因长期使用拓扑异构酶毒剂而出现的继发性急性髓样白血病以及心脏毒性。因此,开发安全,高效,低毒以及可逆转多药耐药的以拓扑异构酶为靶点的化疗药物意义重大。RiccardinD是从苔藓类植物Dumortiera hirsuta分离所得的一种新型的大环双联卞类化合物。前期研究表明,RiccardinD可以抑制真菌被膜的形成,它的类似物羽苔素E可以逆转真菌耐药和肿瘤耐药,另一类似物地钱素C可以通过诱导微管解聚进而导致细胞凋亡最终发挥抗肿瘤作用,还可将肿瘤细胞阻滞在G2/M期抑制其增殖。本研究是基于前期研究结果推测RiccardinD可能会具有抗肿瘤及逆转多药耐药作用。试验方法1. RiccardinD对白血病细胞凋亡作用研究选用人慢性骨髓性白血病细胞株K562及人急性粒细胞白血病细胞株HL60,MTT法检测不同浓度riccardinD对白血病细胞增殖的抑制作用,Hoechst33258, FITC-Annexin V双染法及DNA Ladder检测riccardinD对白血病细胞凋亡的影响Western blotting检测riccardinD对凋亡蛋白Caspase-3, Caspase-9, cleaved PARP9表达的影响。RT-PCR检测riccardinD对细胞凋亡抑制因子Bcl-2与细胞凋亡促进因子Bax-a比值的影响。最后检测线粒体和死亡受体介导的两条凋亡通路中的关键分子Cytochrome c及Caspase-8分析riccardinD可能介导的凋亡通路。2. RiccardinD对拓扑异构酶Ⅱ活性的影响以pBR322 DNA为作用底物,etoposide为阳性对照,检测riccardinD对拓扑异构酶Ⅰ和拓扑异构酶Ⅱ活性的影响。将riccardinD作用于K562细胞后,提取其拓扑异构酶Ⅱ,检测riccardinD对细胞内拓扑异构酶Ⅱ活性的影响。实时定量RT-PCR检测riccardin D对DNA拓扑异构酶Ⅱα基因表达的影响。SPR分析进一步检测riccardinD与拓扑异构酶Ⅱα间的结合能力。选用拓扑异构酶Ⅱα缺陷型细胞株HL60/MX2及构建拓扑异构酶Ⅱα高表达细胞系K562/TopoIIa检测riccardinD对两种细胞增殖的影响。3. RiccardinD对非小细胞肺癌生长的影响体外法,选取非小细胞肺癌细胞H460及A549,采用MTT, Hoechst33258及FITC-Annexin V双染法检测riccardinD对非小细胞肺癌细胞凋亡的影响。另外,划痕试验、重组基底膜侵袭与转移及明胶酶活性测定试验检测riccardinD对非小细胞肺癌细胞侵袭及转移能力的影响。体内法,以H460细胞接种裸鼠腋下,待肿瘤长至100mm3-300mm3开始尾静脉注射给予riccardinD,三天一次,给药三周。给药结束后,取瘤组织称重,计算抑瘤率。取瘤块进行Western blotting及TUNEL试验检测riccardinD体内抑瘤作用及对肿瘤组织内凋亡蛋白Caspase-3, Caspase-9,cleaved PARP及侵袭转移相关蛋白MMP-2, MMP-9, VEGF及Erk1/2的影响。4. RiccardinD对肿瘤多药耐药的影响选用两株耐药株K562/A02及H460/RT,分别是阿霉素和紫杉醇耐药株。采用MTT法检测riccardinD单用,阿霉素与riccardinD合用,紫杉醇和riccardinD合用后对耐药细胞的抑制作用,计算合用后药物逆转倍数。以逆转作用较强的紫杉醇和riccardinD合用组进行裸鼠试验,以H460/RT细胞接种裸鼠腋下,待肿瘤生长至100mm3-300mm3时给予riccardinD和紫杉醇。给药结束,取瘤组织称重,计算抑瘤率。取瘤组织检测凋亡蛋白及耐药蛋白表达。另外,检测riccardinD对K562/A02细胞凋亡蛋白,耐药蛋白及基因表达水平的影响。试验结果1. RiccardinD诱导白血病细胞凋亡MTT结果显示,以riccardin D(10μM-60μM)分别与K562,HL60细胞孵育24h,48h,72h,最高抑制率90.6%。Hoechst33258结果显示,10μM,20μM和30μM riccardin D明显诱导细胞凋亡,孵育48h,细胞核染色质浓染,固缩或集聚核膜周边、或呈块状碎裂改变。FITC-Annexin V双染结果显示,10μM riccardinD处理K562细胞24h凋亡率为25.55%,HL60凋亡率为27.56%。DNA Ladder结果显示,riccardin D处理后产生明显DNA梯状片段。Western blot结果显示,10μM-40μM riccardin D显著增加K562和HL60细胞的Caspase-3, Caspase-9, cleaved PARP蛋白表达水平。RT-PCR显示,riccardin D可显著降低Bcl-2 mRNA水平,增加Bax-a mRNA水平,Bcl-2/Bax-a比值明显降低。此外,10μM-40μM riccardin D可显著增加K562细胞内Cytochrome c释放,而对procaspase-8蛋白表达水平无明显影响。2. Riccardin D靶向拓扑异构酶Ⅱ诱导凋亡拓扑异构酶活性检测显示,riccardin D对拓扑异构酶Ⅰ活性无明显影响,而200μM-800μM riccardin D体外可明显抑制拓扑异构酶Ⅱ活性,呈现浓度剂量依赖关系,而且riccardin D对拓扑异构酶Ⅱ活性的抑制作用明显强于阳性对照etoposide。Riccardin D孵育后的K562细胞内拓扑异构酶Ⅱ活性显著降低。实时定量RT-PCR检测结果表明,riccardin D显著降低K562细胞内DNA拓扑异构酶ⅡαmRNA水平。以SPR技术分析显示,riccardin D与拓扑异构酶Ⅱ高度结合,结合常数KD为2.68E-06。进一步实验,riccardin D对高表达拓扑异构酶Ⅱ细胞抑制作用显著,而对拓扑异构酶Ⅱ缺陷型细胞株HL60/MX2抑制作用较弱,说明riccardin D可能通过靶向拓扑异构酶Ⅱ产生抗癌作用。3.Riccardin D对非小细胞肺癌抑制作用MTT, Hoechst33258, FITC-Annexin V双染及Western blot试验结果均显示,riccardin D显著抑制非小细胞肺癌细胞增殖,促进细胞凋亡及增加凋亡蛋白表达水平。划痕试验结果表明,5μM-10μM riccardin D抑制A549细胞向无细胞划痕区迁移。Transwell侵袭试验显示,riccardin D抑制A549细胞穿透人工基底膜,当riccardin D浓度为5μM, 10μM,20μM时,抑制率分别为40.7%,56.5%,65.4%。明胶酶谱结果显示,2.5μM,5μM,10μM riccardin D显著抑制A549和H460细胞培养液中MMP-2, MMP-9活性。裸鼠移植瘤试验显示,riccardinD明显抑制肿瘤组织生长,20mg/kg时抑制率为44.5%,且裸鼠体重下降不明显。进一步检查肿瘤组织,显示TUNEL阳性染色水平增加,凋亡率为36.9%。4. Riccardin D逆转多药耐药作用以10μM riccardin D与阿霉素联合处理K562/A02可逆转阿霉素耐药,其逆转倍数为2.31。以10μM riccardin D与紫杉醇联合处理H460/RT可逆转紫杉醇耐药,逆转倍数为8.42。Riccardin D与紫杉醇联合处理接种H460/RT的裸鼠,riccardinD,紫杉醇单用组抑瘤率分别为46.8%和48.1%。而riccardin D与紫杉醇合用组肿瘤抑制率为57.3%。与单用组比较,合用组较单用组抑瘤率升高,且凋亡蛋白表达水平明显升高,而耐药蛋白表达水平显著降低。电泳结果显示,riccardin D降低K562/A02细胞内P-gp蛋白及Mdrl基因表达水平。试验结论Riccardin D具有较强的抗癌活性,显示抗侵袭转移活性和逆转多药耐药活性,抑制接种裸鼠的人肺癌H460肿瘤生长。其机制可能通过靶向拓扑异构酶Ⅱ,从而产生对拓扑异构酶Ⅱ活性的抑制作用,启动线粒体介导的凋亡通路,诱导肿瘤细胞凋亡。更多还原

【Abstract】 BackgroundsTopoisomerase is a kind of ribozyme which is essential for the survival of eukaryote and can mediate the single-stranded or double-stranded DNA instantaneous breaks. Topoisomerase plays an important role in DNA replication, transcription, recombination and chromosome segregation. On the basis of different ways of DNA breaks, topoisomerase can be divided into topoisomerasel and topoisomerasell. To interfer with DNA replication by targeting topoisomerase have a predictive value in the adjuvant therapy of malignant tumor. Topoisomerases are the targets of many clinically useful anticancer drugs. But still some problems were shown in the application process such as the generation of multi-drug resistance, the emergence of secondary acute myeloid leukemia due to the long-term use of Topo agents, cardiotoxicity. Therefore, it is of great significance to develope safe, efficient, low toxicity drugs targeting topoisomerase which can reverse multidrug resistance.RiccardinD, a novel macrocyclic bisbibenzyl compound, was extracted from the Chinese liverwort plant Dumortiera hirsutafrom. Previous studies have shown that riccardinD can inhibit the formation of the biofilms of Candida albicans, and its analogues Plagiochin E can reverse fungi multidrug resistance and tumor multidrug resistance, and the other analogues Marchantin C can lead to tumor cell apoptosis by microtubule depolymerization. Marchantin C also can arrest tumor cells in G2/M phase.The preliminary studies suggested that RiccardinD may have anti-tumor effect and reversal of multidrug resistance. The mechanism studies are underway. Methods1. The effects of riccardinD on leukemia cell apopotosisHuman chronic myelogenous leukemia cell line K562 and human acute myelogenous leukemia cell line HL60 were obtained for the detection of apoptosis using Hoechst33258, FITC-Annexin V staining and DNA Ladder detection. Caspase-3, Caspase-9, cleaved PARP expression were determined by Western blotting. RT-PCR was employed to estimate the ratio between anti-apoptosis factor Bcl-2 and pro-apoptosis factor Bax-a. Finally, the two key moleculars cytochromec and caspase8 in the mitochondrial and death receptor-mediated apoptosis pathways were detected by western blotting.2. The effects of riccardinD on topoisomerasesⅡactivityThe effects of riccardinD on topoisomerase I and topoisomeraseⅡactivity were tested with pBR322 DNA as substrate and etoposide as a positive control. The ativity of topoisomerasell which was extracted from K562 cells after treatment with riccardinD were detected. Real-time quantitative PCR was employed to detect the topoisomerasella gene levels. SPR analysis was used to evaluate the binding affinity between riccardinD and topoisomeraseⅡα. TopoisomeraseⅡα-deficient cell lines HL60/MX2 and topoisomerasella-overexpression cell lines were used to detect the effects of riccardinD on cell proliferation.3. The effects of riccardinD on the growth of human non-small cell lung cancer in vitro and in vivoTo test the effects of riccardinD on non-small cell lung cancer cell apoptosis in vitro using MTT, Hoechst33258 and FITC-Annexin V double staining. In addition, the scratch assay, transwell chamber assay and gelatin zymography activity assay were employed to investigate the effects of riccardinD on the anti-invasion and metastasis ability of non-small cell lung cancer cell. Nude mice inoculated with H460 cells were administrated riccardinD when the tumor grew to 100mm3-300mm3. Administration was performed every 3 days for three consecutive weeks. The rates of tumor growth inhibition were defined as a ratio to the vehicle tumor weight. The specimens were removed for TUNEL staining and Western blot analysis for the apoptosis protein Caspase-3, Caspase-9 and cleaved PARP and invasion and metastasis associated protein MMP-2, MMP-9, VEGF, and Erkl/2.4. The effects of riccardinD on the multi-drug resistanceTo test the effects of riccardinD alone, doxorubicin alone and paclitaxel alone on the cell proliferation of K562/A02 named doxorubicin resistant strain and H460/RT named paclitaxel resistant strain using MTT assay. The inhibitory effects of the combination of doxorubicin, paclitaxel and riccardinD were evaluated respectively and the reversal fold was calculated. The nude mice inoculated with H460 cells were administrated to the more effective combination of paclitaxel and riccardinD when the tumor grew to 100mm3-300mm3. The rates of tumor growth inhibition for the combination group and alone were defined as a ratio to the vehicle tumor weight. The specimens were removed for Western blot analysis for the apoptosis protein and multi-drug resistance protein. The effects of riccardinD on multi-drug resistance protein and gene expression were detectd in K562/A02 cells.Results1. RiccardinD induced apoptosis of human leukemia cellsMTT results showed that riccardin D (10μM-60μM) significantly inhibited the proliferation of K562 and HL60 cells after 24h,48h and 72h treatment. The maximum inhibition rate was 90.6%. Hoechst33258 staining results showed that 10μM,20μM, 30μM RiccardinD induced the nuclear chromatin, condensation, or gathering around to the nuclear membrane, or in blocks fragmentation change role in leukemia cells after 48h treatment. FITC-AnnexinⅤdouble staining showed that apoptosis rates were 25.55% and 27.56% after treatment with 10μM riccardin D in K562 and HL60 cells, respectively. DNA Ladder results showed the formation of DNA ladder fragments after treatment with different concentrations of riccardin D with the dose-dependence. Western blot results showed that 10μM-40μM Riccardin D can significantly increase the expression of Caspase-3, Caspase-9, cleaved PARP protein in K562 and HL60 cells. RT-PCR showed the Bcl-2/Bax-αratio was significantly decreased. In addition,10μM-40μM Riccardin D can significantly increase the release of K562 cells cytochrome c with no significant effect on the expression of procaspase8. The activity form was not detected.2. RiccardinD induced apoptosis by targeting DNA topoisomeraseⅡTopoisomerase activity testing results show that riccardin D had no effect on topoisomeraseⅠactivity.200uM-800μM riccardin D inhibited the topoisomeraseⅡactivity significantly in a dose dependent manner. And the inhibitory effects of riccardin D on topoisomeraseⅡactivity were significantly stronger than the positive control etoposide. The topoisomeraseⅡactivity in cells treated with riccardin D was also obviously reduced. Real-time quantitative PCR showed ricca rdin D can significantly reduce the DNA levels of topoisomeraseⅡα. SPR analysis showed that riccardin D revealed a high binding affinity for topoisomeraseⅡwith the equilibrium dissociation constant (KD) of 2.68E-06. Riccardin D did not inhibit the proliferation of topoisomeraseⅡαdeficient cell lines HL60/MX2 with the highest rate 30%. Apoptosis induction was not obvious, either. Compared with the normal K562 cells, riccardin inhibited more significantly for topoisomeraseⅡ-overexpression cell lines.3. Riccardin D inhibited the growth of human non-small cell lung cancer in vitro and in vivoMTT, Hoechst33258, FITC-Annexin V staining and Western blotting results showed that, riccardin D inhibited the cell proliferation of non-small cell lung cancer cells and induced apoptosis and increased the expression of apoptosis protein in vitro. Scratch assay results showed that the 5μM-10μM riccardin D inhibited A549 cells to the migration of cell-free scratch area. Transwell chamber assay showed that at the range of 2.5μM,5μM and 10μM of riccardin D, the number of cells penetrating the Matrigel-coated polycarbonate filters were inhibited by 40.7%,56.5% and 65.4%, respectively. Gelatin zymography results showed the MMP-2, MMP-9 activity was markedly inhibited in the supernatants of A549 and H460 cells exposed to riccardin D. 20 mg/kg of riccardin D delayed the growth of H460 xenografts by 44.5%. Etoposide (20 mg/kg), the positive control drug, inhibited the growth of H460 xenografts by 59.7%. Riccardin D treatment was generally well tolerated by mice with no significant loss of body weight. While, a markedly decrease of body weight was measured in etoposide treated-animals TUNEL staining of tumor tissue showed the cells in riccardinD-treated group were positive staining with blue nuclei. The apoptosis rate was 36.9%.4. Riccardin D reversed the multi-drug resistanceThe combined treatment with 10μM Riccardin D and doxorubicin in K562/A02 reversed the doxorubicin resistance. The reversal fold was 2.31. The combined treatment with 10μM Riccardin D and paclitaxel in H460/RT reversed the paclitaxel resistance. The reversal fold was 8.42. Riccardin D alone, paclitaxel alone and the combination treatment in nude mice inoculated H460/RT cells were evaluated. The inhibition rates were 46.8%,48.1% for alone group. The tumor inhibition rate was 57.3% in the combined group with paclitaxel. Electrophoresis showed that the apoptosis protein in tumor tissue of combination group was significantly increased. Riccardin D significantly reduced P-gp protein and the gene expression Mdrl in K562/A02 cells.ConclusionRiccardin D inhibited the tumor growth in nude mice inoculated H460 cells significanty, and also had the ability of anti-invasion and metastasis and reversed the multidrug resistance.The mechanisms were involved in targeting topoisomerasell. Riccardin D revealed a high binding affinity with topoisomerasell and obviously inhibited the topoisomerasell activity. Finally, riccardinD induced apoptosis of leukemia cells through the mitochondrial mediated apoptosis pathway.更多还原