【作者】 李娟；

【导师】 刘培淑；

【作者基本信息】 山东大学 ， 妇产科学， 2009， 博士

【摘要】 研究背景:大黄素(Emodin)属蒽醌类衍生物,是大黄、虎杖、首乌等多种中药的有效成分之一,除了具有调节机体免疫,抗菌抗炎等作用外,近年来研究表明,大黄素还具有诱导肿瘤细胞凋亡及抑制肿瘤细胞转移等多种生物学效应。随着研究的逐步深入,其抗肿瘤作用越来越受到人们的重视。卵巢癌(Ovarian cancer,OC)是妇科中的常见恶性肿瘤,其死亡率居妇科肿瘤首位。手术治疗和以紫杉醇、顺铂等为主的联合化疗方案是目前卵巢癌治疗的重要手段。而在临床治疗过程中会出现由于长期治疗而导致肿瘤细胞对化疗药物产生耐药性,约80%以上的卵巢癌患者在治疗后会出现肿瘤病灶的侵袭和转移,从而导致肿瘤复发,五年存活率仅45%。如何逆转卵巢癌细胞的耐药及抑制卵巢癌侵袭转移等问题成为卵巢癌治疗的热点和难点。紫杉醇(Paclitaxel)是卵巢癌化疗的一线药物,作用机理是诱导和促进微管的装配,使微管在有丝分裂时不能形成纺锤体和纺锤体丝,导致快速分裂的肿瘤细胞在有丝分裂阶段(G2/M期阻滞)固定,阻止肿瘤细胞的分裂和繁殖,诱导细胞凋亡。但在经过数个疗程化疗后,肿瘤细胞往往会出现对紫杉醇的获得性耐药,致使后续化疗失败。紫杉醇耐药的机制包括P-gp(p-glycoprotein,P-gp)表达的升高,微管α、β亚基的改变,凋亡途径改变等。P-gp是多药耐药基因-1(multidrug resistance gene-1,MDR-1)编码的一种跨膜转运蛋白,具有ATP酶活性,能将化疗药物从细胞内泵出,降低细胞内的有效药物浓度从而导致多药耐药,其过表达是紫杉醇耐药的重要原因之一。凋亡抑制蛋白家族(Inhibitor ofapoptosis proteins,IAPs)中的survivin、X染色体连锁凋亡抑制蛋白(X-linkedinhibitor of apoptosi s protein,XIAP)在凋亡过程中能够发挥重要的抑制细胞凋亡的作用,其过表达是导致紫杉醇耐药的机制之一。巨噬细胞移动抑制因子(macrophage migration inhibitory factor,MIF)是一种重要的免疫及神经内分泌因子,具有信号调节、免疫活性、促进炎症反应、催化功能以及内分泌功能等多重生物学活性。它还可以刺激细胞增殖、分化和迁移,抑制肿瘤细胞的凋亡,促进肿瘤血管生成,在多种肿瘤的发生、发展和转移中担任重要角色。MIF蛋白在卵巢癌、食管癌、肝细胞癌等多种肿瘤中的表达显著升高,表明MIF参与了肿瘤的发生发展过程。MIF能通过调控细胞外信号调节激酶(ERK)调节的MAPK的激活和磷酸化来促进肿瘤细胞的增殖;通过上调肿瘤MMP-9和MMP-2的表达,增加对肿瘤细胞外基质以及基底膜的溶解;使肿瘤细胞中VEGF、IL-8的表达升高,促进肿瘤细胞转移过程中微血管的形成,这些可能是MIF促进肿瘤细胞侵袭和远处转移的重要因素。基质金属蛋白酶(matrixmetaloproteinases,MMPs)是一族锌肽酶超家族,参与肿瘤细胞对基底膜(basement membrane,BM)和细胞外基质(extracellular matrix,ECM)的降解,是目前已知的能够降解胶原纤维的唯一酶类。MMPs在多种转移癌中表达明显增高,与肿瘤转移密切相关。MMPS成员中,基质金属蛋白酶-2,-9(matrixmetalloproteinase-2,-9,MMP-2,-9)因为能够特异性的降解细胞外基质和基底膜的主要成Ⅳ型胶原,从而在肿瘤侵袭转移过程中发挥最直接和最重要的作用。本研究分为两部分:第一部分:大黄素对卵巢癌细胞系诱导凋亡和逆转耐药作用及机制的研究;第二部分:大黄素对卵巢癌耐药细胞系侵袭能力的抑制作用及其机制研究。第一部分:大黄素对卵巢癌细胞系诱导凋亡和逆转耐药作用及其机制的研究目的:1.研究大黄素(Emodin)作用于人卵巢癌细胞系A2780和A2780/taxol细胞的生长抑制作用;2.研究大黄素对卵巢癌细胞的凋亡诱导作用;观察大黄素对紫杉醇诱导耐药细胞系A2780/taxol凋亡的变化;3.检测大黄素作用前后卵巢癌细胞中耐药及凋亡相关基因及蛋白的表达水平,探讨大黄素的作用机制。方法:MTT法检测不同浓度大黄素(10,20,40,80μM)对A2780和A2780/taxol的生长抑制作用;MTT法检测低浓度大黄素联合紫杉醇作用时,对紫杉醇抑制耐药卵巢癌细胞A2780/taxol生长的增强作用;流式细胞仪(FCM)检测大黄素单独或联合紫杉醇作用于耐药卵巢癌细胞A2780/taxol的凋亡诱导情况;QRT-PCR检测大黄素作用后A2780/taxol细胞内MDR-1、XIAP和survivin基因mRNA表达;蛋白印迹法(Western blot)检测A2780和A2780/taxol细胞中MDR-1、XIAP和survivin蛋白的表达,并检测大黄素作用后A2780/taxol细胞中MDR-1、XIAP和survivin蛋白表达;罗丹明外排实验检测P-gp功能水平的改变。结果:1、大黄素对A2780和A2780/taxol细胞的生长抑制作用应用MTT法检测不同浓度大黄素作用于卵巢癌细胞系A2780和A2780/taxol24小时后的生长抑制作用,结果显示:A2780细胞各组细胞增殖率分别为:空白对照组99.81%±3.43%;大黄素10μM组93.85%±4.21%;大黄素20μM组78.17%±1.26%;大黄素40μM组55.90%±7.00%;大黄素80μM组38.91%±3.68%。A2780/taxol细胞各组细胞增殖率分别为:空白对照组98.63%±4.61%:大黄素10μM组93.10%±5.13%;大黄素20μM组75.61%±3.82%;大黄素40μM组57.76%±6.30%;大黄素80μM组36.21%±4.50%。2、大黄素对A2780和A2780/taxol细胞的凋亡诱导作用应用FCM法检测不同浓度大黄素作用于卵巢癌细胞系A2780/taxol 24小时后的凋亡诱导作用,结果显示各组细胞凋亡率分别为:空白对照组1.30%;10μM大黄素组5.56%;20μM大黄素组17.66%;40μM大黄素组31.96%;80μM大黄素组41.97%。3、大黄素与紫杉醇对A2780和A2780/taxol细胞的联合作用应用MTT法检测大黄素(10μM)和紫杉醇(1μM)单独或联合作用于卵巢癌细胞系A2780和A2780/taxol 24小时后的生长抑制作用,结果显示,A2780细胞各组细胞增殖率分别为:空白对照组100.00%±3.23%;大黄素10μM组95.00%±3.86%;紫杉醇1μM组56.45%±2.93%;大黄素+紫杉醇组43.29%±2.43%。细胞各组细胞增殖率分别为:空白对照组98.80%±3.17%;大黄素10μM组92.34%±1.07%;紫杉醇1μM组81.49%±4.49%;大黄素+紫杉醇组63.60%±2.27%。4、A2780和A2780/taxol细胞中MDR-1、XIAP和survivin的基础表达应用Western blot法检测处于对数生长期的A2780和A2780/taxol细胞中MDR-1、XIAP和survivin的基础表达,结果显示:MDR-1所编码的P-gp蛋白的表达在A2780细胞中非常低,但在A2780/taxol细胞中表达明显升高。A2780/taxol细胞中,凋亡抑制蛋白家族的survivin和XIAP的表达也比在其亲本细胞A2780中表达明显升高。5、大黄素对A2780/taxol细胞中MDR-1、XIAP和survivin基因及蛋白表达水平的影响应用QRT-PCR和Western blot法检测大黄素作用后A2780/taxol细胞中MDR-1、XIAP和survivin的表达水平,QRT-PCR结果用目的基因和内参照基因(β-actin)的比值来表示。结果显示:空白对照A2780/taxol细胞中MDR-1基因水平为1.20±0.25,大黄素10μM干预72小时后A2780/taxol细胞中MDR-1基因水平为0.27±0.09(P＜0.05);空白对照A2780/taxol细胞中XIAP基因水平为1.77±0.41,大黄素10μM干预72小时后A2780/taxol细胞中XIAP基因水平为0.987±0.20(P＜0.05);空白对照A2780/taxol细胞中survivin基因水平为0.086±0.01,大黄素10μM干预72小时后A2780/taxol细胞中survivin基因水平为0.06±0.01(P＜0.05)。Western blot的结果显示大黄素作用后MDR-1、XIAP和survivin蛋白的表达水平降低。罗丹明实验表明大黄素作用后A2780/taxol细胞中P-gp蛋白的功能为空白对照细胞的32.11%±8.8%(P＜0.05)。结论:1、大黄素可以抑制A2780和A2780/taxol细胞的增殖,具有浓度依赖性;2、大黄素能诱导A2780和A2780/taxol细胞凋亡,通过降低MDR-1、XIAP和survivin的表达,增强紫杉醇耐药细胞对紫杉醇的药物敏感性并促进紫杉醇诱导的细胞凋亡。第二部分大黄素对卵巢癌耐药细胞系侵袭能力的抑制作用及其机制研究目的:1、探讨A2780/taxol细胞侵袭能力的改变及相关机制;2、检测大黄素对卵巢癌A2780/taxol细胞的侵袭抑制作用;并探讨大黄素抑制肿瘤细胞侵袭能力的作用机制。方法:肿瘤细胞侵袭实验(Transwell)检测A2780和A2780/taxol两种细胞的侵袭能力;Western blot法检测侵袭转移相关因子MIF、MMP-2和MMP-9在A2780和A2780/taxol细胞中基础表达的差异;Transwell法检测不同浓度大黄素(20,40,80μM)作用后卵巢癌A2780/taxol细胞体外侵袭能力的改变:QRT-PCR法检测大黄素作用前后A2780/taxol细胞中MIF、MMP-2和MMP-9基因mRNA表达水平的变化;Western-blot法检测大黄素作用前后A2780/taxol细胞中MIF、MMP-2和MMP-9蛋白表达水平的变化。结果:1、A2780和A2780/taxol细胞的侵袭能力的检测应用Transwell法检测处于对数生长期的A2780和A2780/taxol细胞的侵袭能力,结果显示:每高倍镜视野中有48±4.64个A2780细胞穿透基底膜层,进入侵袭小室下层;每高倍镜视野有92.33±7.40个细胞穿透基底膜,进入侵袭小室下层,差异有统计学意义(P＜0.05)。2、A2780和A2780/taxol细胞中MIF、MMP-2和MMP-9的表达水平应用western blot法检测出于对数生长期的A2780和A2780/taxol细胞中MIF、MMP-2和MMP-9蛋白的基础表达,结果显示:A2780/taxol细胞中侵袭相关蛋白MIF、MMP-2和MMP-9的表达明显高于其亲本细胞系A2780;3、大黄素对卵巢癌A2780/taxol细胞侵袭能力的影响应用Transwell法检测不同浓度大黄素作用24小时后卵巢癌A2780/taxol细胞的侵袭能力,结果显示:空白对照组有96.33±11.82个细胞穿透基底膜,进入侵袭小室下层;20μM组有58.0±8.47个细胞进入侵袭小室下层;40μM组有33.44±6.97个细胞进入侵袭小室下层;80μM组有16.22±7.36个细胞进入侵袭小室下层。4、大黄素作用于卵巢癌A2780/taxol细胞后MIF、MMP-2和MMP-9的表达应用ORT-PCR和western blot法检测不同浓度大黄素作用于A2780/taxol细胞24小时后细胞中MIF、MMP-2和MMP-9基因和蛋白的表达水平。QRT-PCR结果用目的基因和内参照基因(β-actin)的比值来表示。结果显示:空白对照A2780/taxol细胞中MIF mRNA水平为1.57±0.16;大黄素20μM干预24小时后,A2780/taxol细胞中MIF mRNA水平为1.35±0.18;大黄素40μM作用后,A2780/taxol细胞中MIFmRNA水平为0.95±0.15;大黄素80μM作用后A2780/taxol细胞中MIF mRNA水平为0.44±0.08。空白对照A2780/taxol细胞中MMP-2 mRNA水平为0.73±0.06;大黄素20μM干预24小时后,A2780/taxol细胞中MMP-2基因水平为0.62±0.08;大黄素40μM作用后MMP-2 mRNA水平为0.27±0.08;大黄素80μM作用后MMP-2 mRNA水平为0.14±0.05。空白对照A2780/taxol细胞中MMP-9 mRNA水平为1.00±0.05;大黄素20μM干预24小时后,A2780/taxol细胞中MMP-9基因水平为0.85±0.07;大黄素40μM作用后MMP-9 mRNA水平为0.58±0.04;大黄素80μM作用后MMP-9mRNA水平为0.38±0.06。Western blot的结果显示大黄素作用后MIF、MMP-2和MMP-9蛋白的表达水平降低。结论:1、卵巢癌细胞A2780/taxol的侵袭能力比A2780显著增强,与MIF、MMP-2和MMP-9的表达升高有关;2、大黄素能够抑制A2780/taxol细胞的体外侵袭能力,具有浓度依赖性;MIF、MMP-2和MMP-9参与了大黄素对卵巢癌细胞侵袭能力的调节。更多还原

【Abstract】 Backgroud:Emodin is an effective component of traditional Chinese herbal medicine. It can be extracted from Rheum, Polygonum cuspidatum, Polygonum multiflorum, et al. Emodin has long been known to have a number of biological activities, such as anti-bacterial, immunosuppressive and anti-inflammatory effects, but its capability of anti-cancer and anti-invasion has not been adequately studied. Ovarian cancer has the highest mortality rate among gynecologic malignancies in the world. The standard treatment for ovarian cancer remains surgical debulking and chemotherapy with carboplatin and paclitaxel. However, the majority of ovarian cancer patients have one of the lowest 5-year survival rates, and the development of drug resistance is a major impediment toward successful treatment of the cancer. Thus, there have been a number of researches aimed at understanding the mechanisms of drug resistance, so as to develop strategies to overcome the disease.Paclitaxel is an effective chemotherapeutic agent and widely used for the treatment of ovarian cancer, whereas the development of drug resistance limits its availability. To date, several mechanisms of paclitaxel resistance in ovarian cancer cells have been proposed, including overexpression of ABC/MDR transporter family of proteins to increase the cellular efflux of paclitaxel, delayed G2/M transition, and alterations in apoptosis regulation. The overexpression of P-gp (P-glycoprotein), which is encoded by MDR-1 (Multidrug resistance-1) gene, plays a key role in the resistant mechanisms. P-gp functions as an efflux pump to decrease the intracellular accumulation of a variety of lipophilic drugs, including paclitaxel, leading to decresed therapeutic efficacy. Successful treatment with chemotherapeutic agents is largely dependent on their ability to trigger cell death in tumor cells; therefore, try to decrease the over-expression of anti-apoptotic molecules may affect the proximal level of apoptotic threshold. As direct caspase inhibitors and participants in a variety of survival signaling pathways, the inhibitor of apoptosis protein (IAP) family are important to the control of cell proliferation and drug resistance in multiple cancer types. As the important IAPs members, XIAP (X-linked inhibitor of apoptosis protein) and survivin up-regulated in most human tumor cells and make the caner cells escape from apoptosis. Evidence indicates that survivin and XIAP are correlated with chemresistance and decrease of these IAPs could induce apoptosis in chemoresistant human ovarian cancer cells. Down regulation of XIAP and survivin may enhance apoptosis in ovarian cancer cells.MIF (macrophage migration inhibitory factor, MIF) was one of the first cytokine activities to be described, originally being identified as a product of activated T lymphocytes that inhibited the random migration of cultured macrophages. Recently, MIF has also been implicated in many processes associated with tumor survival and invasion, such as stimulate cell proliferation, differentiation and migration, promote tumor angiogenesis. MIF expression is increased during the evolution of several malignancies, breast-, prostate-, colon-, lung- and ovarian-derived tumors have all been shown to contain significantly higher levels of MIF message and protein than their noncancerous cell counterparts. MIF has been reported to promote angiogenesis and the invasiveness and matrix degrading activity of cancer cells by stimulating mmp-2 and mmp-9. Taken together, MIF may be an important connective point in the process during invasive inhibition of emodin.MMPs (matrix metaloproteinases, MMPs), inuclding MMP-2 and MMP-9, play an important role in the degradation of basement membrane type IV collagen, which is associated with tumor cell invasion and metastasis. A role for MMPs in ovarian cancer development has been postulated based upon the observation that several members of the MMP family are up-regulated during ovarian cancer neoplastic progression. MMPs may play important role in the invasion and migration of tumor cells.Part I Emodin sensitizes human ovarian cancer cells topaclitaxel-induced apoptosisObjective:Ovarian cancer has the highest mortality rate among gynecologic malignancies in the world, and the development of drug resistance is a major impediment toward successful treatment of the disease. Emodin has been reported to sensitize human tumor cells to chemotherapeutic agents. In present study, whether emodin could overcome chemoresistance of paclitaxel resistant A2780/taxol cells and the mechanism were investigated.Methods:Cells were incubated and then treated with different concentration of emodin (10, 20, 40, 80μM) for 24h, respectively, then the viability of each group was determined by MTT assays. The apoptosis rate of cells treated with emodin or/and paclitaxel was determined by FCM. The expression of gene and protein level of MDR-1、XIAP and survivin were detected by QRT-PCR and western blot respectively. The function of P-gp was determined by FCM.Results:We found that emodin could induce apoptosis at a high concentration and enhance apoptosis induced by paclitaxel at a low concentration in both paclitaxel sensitive and resisitant ovarian cancer cell lines. QRT-PCR and western blot results indicated that the MDR-1 gene and P-gp, which was encoded by MDR-1 gene, were decreased after treatment with emodin. The function of P-gp is also attenuate. Meanwhile, the expression of XIAP and survivin were significantly down-regulated in cells treated with emodin. Furthermore, we found emodin could enhance the expression of cleaved fragments of caspase-3.Conclusion:Emodin could induce apoptosis alone and sensitize ovarian cancer cells to paclitaxel induced apoptosis. The results demonstrated that emodin could induce apoptosis of resistant ovarian cancer cells by increasing the cellular concentration of paclitaxel and decreasing the expression of anti-apoptotic molecules.Part II Emodin inhibits invasion activity of ovarian cancer cellsObjective:Ovarian cancer has the highest mortality rate among gynecologic malignancies in the world. Most of patients die of tumor invasion. Emodin has been reported to inhibit invasion activity in human tumor cells. In present study, whether emodin could inhibit cell invasion ability of paclitaxel resistant ovarian cancer cells were investigated.Methods:We checked the invasion ability of different ovarian cancer cells, and then checked the expression of invasion related factors MIF, MMP-2 and MMP-9 in A2780 and A2780/taxol cells. Then the high invasiveness cells were treated with different concentration of emodin (20, 40, 80 u M), then the cell invasion ability was measured by transwell assays. The changes of mRNA and protein expression of MIF, MMP-2 and MMP-9 were examined by QRT-PCR and western blot respectively.Results:A2780/taxol cells had high invasiveness than A2780 cells, and the expression of MIF, MMP-2 and MMP-9 were up-regulated in A2780/taxol cells. The invasive potential of both cell lines were reduced dramatically after treatment with emodin. QRT-PCR and western blot results indicated that the gene and protein expression of MIF, MMP-2 and MMP-9 were decreased after treatment with emodin. And the effect of emodin showed dose-dependent manner in ovarian cancer cells.Conclusion:A2780/taxol cells is more invasiveness than A2780 cells. The possible mechanisms are increased MIF, MMP-2 and MMP-9. Emodin could inhibit tumor invasion ability by down-regulation the expression of MIF, MMP-2 and MMP-9 in ovarian cancer cells.更多还原