【作者】 王丹；

【导师】 惠宁；

【作者基本信息】 第二军医大学 ， 妇产科学， 2011， 博士

【摘要】 上皮性卵巢癌(epithelial ovarian cancer,EOC)是严重威胁妇女健康的疾病,发病率在妇科恶性肿瘤中已上升至第二位,死亡率居于第一。大约70%的卵巢癌患者为Ⅲ期或Ⅳ期病变。肿瘤细胞减灭术+铂类为基础的联合化疗是卵巢癌公认的最佳初治方案,但大约70%～80%达到完全缓解的患者在首次治疗2年内出现肿瘤复发,并且治愈的可能性很小。尽管应用了补救性化疗治疗复发,但是这并不能使总的无瘤生存期和总的生存率延长。复发性卵巢癌患者的预后仍然很差,5年生存率仅为23%,而Ⅲ期和Ⅳ期卵巢癌患者只有14%。主要是因为卵巢癌对化疗药物产生了耐药性。因此对于卵巢癌,尤其是化疗耐药卵巢癌的治疗成为了改善肿瘤预后、提高患者生存率的最关键性因素。流行病学研究显示,甾体激素在卵巢癌的发生发展中起一定的作用,促性腺激素、雌激素、雄激素可能是致病因子,而促性腺激素释放激素和孕酮可能是保护因子。实验研究显示,有些激素的受体在卵巢癌细胞中表达,并介导激素对细胞的生长刺激和生长抑制效应,包括促性腺激素释放激素(gonadotropin-releasing hormone,GnRH)受体、黄体生成素(1uteinizing hormone,LH)受体、卵泡刺激素(follicle stimulating hormone,FSH)受体、雌激素受体、孕激素受体和雄激素受体等。这为开展卵巢癌的内分泌治疗提供了可能。内分泌治疗激素依赖性肿瘤现已广泛用于临床,具有给药方便,毒副作用小,不良反应少,疗效持久等优点,对于乳腺癌、前列腺癌、子宫内膜癌等,内分泌治疗常作为首选的治疗手段。在内分泌治疗的药物中,尤以GnRH类似物的应用备受关注,其在上述肿瘤的治疗中均取得了良好的临床疗效。GnRH为下丘脑分泌的十肽激素,是下丘脑-垂体-性腺轴的关键信号分子,能与垂体前叶促性腺细胞的GnRH受体特异性结合,调节垂体LH、FSH的合成和分泌。LH、FSH释放进入血循环,作用于性腺,调节生殖细胞成熟及性激素合成分泌。“促性腺激素假说”是关于性激素在卵巢癌发病机制中的作用的第一个假说。它推测卵巢癌的发生是LH、FSH对卵巢组织过度刺激的结果。体外研究显示,促性腺激素可能通过FSH受体和/或LH受体的介导,诱导上皮生长因子受体(epidermal growth factor receptor,EGFR)和血管内皮生长因子(vascular endothelial growth factor,VEGF)表达刺激卵巢表面上皮细胞(ovarian surface epithelium,OSE)的增殖,而这些因子对卵巢癌的发生发展起作用。丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)的激活也参与FSH在肿瘤发生前和肿瘤性的OSE细胞的生长刺激效应。目前已发现两种形式的GnRH。GnRH-Ⅰ又称促黄体生成激素释放激素,它不仅在哺乳动物的生殖调节中起关键作用,也是卵巢增殖活性的调节子。通过置换或去除GnRH-Ⅰ第6位和第10位氨基酸,可得到GnRH类似物,其生物学效应较天然GnRH提高50-100倍。在卵巢癌中,GnRH类似物抗肿瘤的可能机制被认为是通过脱敏(GnRH激动剂,GnRH agonist,GnRHa)或下调(GnRH拮抗剂,GnRH antagonist,GnRH-ant)垂体GnRH-Ⅰ受体,导致促性腺激素(FSH,LH)分泌下降和性腺甾体激素减少,而性腺甾体激素是肿瘤生长因子。除了调节促性腺激素及其受体,在体内和体外实验中,GnRH-Ⅰ及其类似物可通过捕获细胞周期和诱导细胞凋亡而减少细胞增殖。GnRH-Ⅰ的抗增殖效应也被认为是由细胞外信号调节激酶1/2(extracellular signal regulated kinase,ERK1/2)途径介导的。另一种形式的GnRH,GnRH-Ⅱ,在鸡的下丘脑中发现,并在脊椎动物中具有结构保守性。约80%的卵巢上皮性肿瘤中表达GnRH-Ⅰ受体,但至今未发现一种功能性的人GnRH-Ⅱ受体转录因子的表达。GnRH类似物的抗增殖作用是通过GnRH-Ⅰ受体介导的。多数有关GnRH激动剂的临床试验[曲普瑞林(Triptorelin)、亮丙瑞林(Leuprolide)、戈舍瑞林(Goserelin)]是在至少对一种化疗方案无效的难治性或复发性卵巢癌患者中进行的。目前的结论是GnRH激动剂可能对复发性卵巢癌患者提供了一种中等有效的补救治疗,甚至在一些病例中可能实现长期持续的疾病稳定。由于应用GnRH激动剂治疗没有明显的副作用,因此,对于不能耐受或接受化疗的患者来说仍然是一种有效的选择。有关GnRH激动剂的实验室研究,尤其是对耐药卵巢癌细胞作用的观察及可能机制的探索,目前国内外尚少见相关报道。本课题首先在体外诱导筛选出对顺铂耐药的卵巢癌细胞亚系,并观察了耐药细胞与亲本细胞之间的生物学特性差异。实验结果显示,伴随卵巢癌细胞对顺铂耐药性的增加,细胞增殖速度减慢,而抗失巢凋亡和侵袭转移能力增加。随后,课题重点观察了GnRH激动剂对耐药卵巢癌的作用并探索其可能的机制。研究结果显示,GnRH激动剂可以抑制耐药卵巢癌细胞的增殖,可与顺铂协同增加顺铂对耐药细胞的毒性,起到化疗增敏作用;而且,GnRH激动剂还能够抑制耐药卵巢癌细胞的侵袭和转移。GnRH激动剂协同顺铂抗耐药卵巢癌细胞的增殖效果在裸鼠体内试验中也得到了的验证。以上结果可能的机制为GnRH激动剂能够抑制EGFR-MAPK-ERK1/2信号通路的激活,并下调由EGF引起基质金属蛋白酶(matrix metalloproteinases,MMPs)的高表达(主要是MMP-2和MMP-9 ),以及上调金属蛋白酶组织抑制剂( tissue inhibitor of metalloproteinases,TIMPs)的表达(主要是TIMP-2和TIMP-1)有关。目的:体外诱导筛选人卵巢癌顺铂(Cisplatin,CDDP)耐药细胞亚系,并继而建立耐药细胞的裸鼠皮下荷瘤模型。方法:采用逐步递增顺铂浓度、间歇作用的方法,体外诱导建立卵巢癌OVCAR-3耐药细胞亚系。取对数生长期的亲本和耐药卵巢癌细胞,MTT法测定细胞的IC50和耐药指数;双层软琼脂集落隆形成实验观察细胞的恶性增殖能力;流式细胞仪技术测定细胞周期的变化和抗失巢凋亡的情况;Transwell小室观察细胞的体外侵袭和转移能力。裸鼠皮下接种耐药卵巢癌细胞建立动物荷瘤模型。结果:(1)成功建立了CDDP耐药细胞亚系模型,命名为OVCAR-3/CDDP细胞,耐药指数测定为13.42;(2)细胞周期测定显示:耐药细胞较亲本细胞增殖缓慢,G0/G1期细胞比例增加,S期细胞比例减少,两者之间有统计学差异(P<0.05);(3)耐药细胞与亲本细胞比较,克隆形成率降低(0.31%±0.07%)vs(0.66%±0.09%),两者之间有统计学差异(P<0.05);(4)Transwell小室实验发现,耐药细胞较亲本细胞侵袭和转移能力增强[(143.6±9.1)vs(95.8±6.2);(233.1±8.5)vs(167.4±5.9);结果均有统计学差异,P<0.01];(5)抗失巢凋亡能力测定显示:耐药细胞较亲本细胞更易聚集,细胞凋亡指数下降[(7.78±1.32)% vs(15.41±1.26)%],比较结果有统计学差异(P<0.01);(6)成功建立了耐药卵巢癌细胞的裸鼠皮下荷瘤模型,成瘤率100%。结论:与OVCAR-3细胞相比,顺铂耐药的OVCAR-3/CDDP细胞增殖能力减弱,但抗失巢凋亡能力和侵袭转移能力均增强。目的:观察GnRH激动剂[D-Trp6] GnRH-I (Triptorelin)对耐药卵巢癌细胞的增殖、细胞周期和细胞凋亡及耐药逆转的作用,并探讨其可能机制。方法:设计Triptorelin、CDDP单药及两者联合药物作用的不同情况下:MTT法观察耐药细胞的增殖抑制情况及计算耐药逆转倍数;流式细胞术评价联合用药对耐药细胞周期和细胞凋亡的作用;免疫印迹法(Western blot)检测ERK1/2信号通路在Triptorelin增敏顺铂细胞毒作用过程中的改变;流式免疫荧光法测定卵巢癌细胞表面EGFR的变化情况;药物作用于耐药细胞裸鼠皮下荷瘤模型,免疫组化法检测移植瘤中EGFR蛋白表达。结果:(1)Triptorelin对耐药卵巢癌细胞的增殖抑制作用呈时间-剂量依赖性;(2)Triptorelin与CDDP联合作用时可增加耐药细胞对CDDP的敏感性,化疗逆转倍数RR为3.91,评价两药联合的效果为协同作用;机制可能是通过Triptorelin能够下调EGFR表达以及抑制ERK1/2的活性;(3)Triptorelin单用和与CDDP联合作用时均可增加耐药细胞周期中G0/G1期细胞的比例,减少S期细胞比例;但Triptorelin单用时细胞周期分布的改变随药物浓度的改变而变化不明显,两药联用时细胞周期的改变随着Triptorelin浓度的增加而明显;(4)CDDP、Triptorelin以及两药联合均有诱导耐药细胞凋亡的作用,以两药联合时作用最强(P<0.01),CDDP、Triptorelin单用时作用依次减弱;(5)Triptorelin可抑制耐药卵巢癌细胞裸鼠皮下移植瘤的生长,与CDDP联合作用时抑制作用较单用时更加明显,P<0.01;(6)免疫组化结果显示,不同药物作用情况下的EGFR蛋白表达在两药联合时下降最明显,P<0.05。结论:体内、体外实验结果均提示,Triptorelin可抑制卵巢癌耐药细胞的增殖,与CDDP联合应用可增加耐药细胞周期中G0/G1期细胞的比例,减少S期细胞比例,并能诱导耐药细胞的凋亡。Triptorelin能够协同顺铂增敏耐药细胞的化疗敏感性,此种作用可能与其下调细胞EGFR的表达及抑制ERK1/2的活性有关。目的:观察GnRH激动剂Triptorelin对耐药卵巢癌细胞体外侵袭和转移的作用,并探讨其可能的分子机制。方法:Transwell小室侵袭和迁移实验观察EGF、AG1478和Triptorelin不同处理情况下,人耐药卵巢癌OVCAR-3/CDDP细胞的侵袭和转移能力的变化情况;ELISA法检测不同药物处理组MMP-2,MMP-9,TIMP-1,TIMP-2的蛋白表达量情况;Western blot蛋白免疫印记法检测EGF,AG1478和Triptorelin对耐药细胞P-EGFR蛋白表达的影响。结果:(1)Transwell小室侵袭和迁移实验显示:不同药物处理情况比较,EGF(10μg/L)提高EGFR活性后能明显地增加耐药细胞的体外侵袭和迁移率(P<0.01),应用AG1478阻断EGFR后能使EGF的促细胞侵袭迁移的作用明显下降,Triptorelin有与AG1478同样的作用效果;(2)ELISA法测定显示:EGF能增加耐药细胞MMP-2,MMP-9蛋白量的表达,减少TIMP-1和TIMP-2蛋白量的表达;而AG1478和Triptorelin均能抑制EGF对OVCAR-3/CDDP细胞的影响,使MMP-2,MMP-9的蛋白表达量下降,TIMP-1和TIMP-2的蛋白表达量上升,比较有统计学差异(P<0.01);但Triptorelin和AG1478的作用之间比较无统计学差异(P>0.05)。结论:Triptorelin能够抑制人卵巢癌耐药细胞的体外侵袭和转移,这可能与Triptorelin抑制EGF诱发的EGFR信号通路的激活,改变MMP-2,MMP-9,TIMP-1和TIMP-2蛋白的表达有关。更多还原

【Abstract】 In China, the incidence of ovarian cancer has increased over the past few decades, while that of cervical cancer, the most common gynecologic cancer, and has decreased. Ovarian carcinoma is most frequently detected when disease has already disseminated intraabdominally, resulting in a 5-year survival rate of less than 20% owing to complications of metastasis. Despite the combination of cytoreductive surgery and platinum-based chemotherapy, most patients in advanced stages eventually relapse and ultimately die because of chemoresistant disease after short periods of responsiveness. Thus, there is a need for new therapeutic targets and a better understanding of the mechanisms involved in the spread of ovarian carcinoma.Recently, the gonadotropin releasing hormone (GnRH) agonist has been used in the treatment of endocrine-dependent tumors. The inhibitory effect of the GnRH agonist on tumor cells was thought to be mediated through a GnRH receptor on the membrane of ovarian, endometrial, and breast cancers. GnRH receptor expression has been shown in 80% of human ovarian tumors and numerous cancer cell lines, and the analogues inhibit proliferation of the GnRH receptor-bearing tumor cells both in vivo and in vitro, supporting evidence for a direct antiproliferative effect. Of particular interest, levels of GnRH receptor seem to be associated with cancer grading and have been reported to be elevated in advanced-stage (stages III and IV) ovarian carcinomas. This opens the possibility that GnRH could directly regulate tumor progression of ovarian cancer cells. Other researchers have also suggested the inhibition of ovarian cancer by the GnRH agonist in experiments. Meanwhile, clinical studies on single GnRH agonist treatment for far advanced ovarian cancer and on combination therapy with cisplatin have demonstrated conflicting results. In order to resolve the differences in results between experimental data and clinical studies, it is necessary to study the mechanism of GnRH agonist as an antitumor regimen for ovarian cancer.The knowledge of MAPK-ERK1/2 involvement in cell response to cisplatin may have important clinical implications in cancer chemotherapy. Nowadays, cisplatin is one of the most effective and commonly used chemotherapeutic for the treatment of ovarian carcinoma. The major limitation for successful cancer chemotherapy is intrinsic or acquired drug resistance of tumor. Matrix metalloproteinases (MMPs) are largely implicated in promoting angiogenesis and tumor metastasis. Above all, MMP-2 and MMP-9 have been suggested to be critical for the invasive and metastatic potential in ovarian carcinoma. MMP activity is often controlled by a specific inhibitor known as the tissue inhibitor of metalloproteinases (TIMP). TIMP-1 selectively binds pro-MMP-9, whereas TIMP-2 associates with pro-MMP-2 in a crucial step in the cell-mediated activation of MMPs (13). By inhibiting active MMPs, TIMPs inhibit cell invasion in vitro and tumorigenesis and metastasis in vivo. Elevated EGFR is associated with less favorable disease outcome in ovarian cancer, related in part to EGFR activation of signaling cascades that lead to enhanced matrix metalloproteinase expression and/or function.The purpose of our study is to explore the potentiating effect of Triptorelin for chemotherapy by down-regulation the activity of ERK1/2, EGFR and/or matrix metalloproteinases in OVCAR-3/CDDP cells ,which are resistant to cisplatin.Objective: The drug-resistant cell line established in vitro is generally used as a research mode1.In this study we designed to establish a Cisplatin- induced human ovarian cancer drug- resistant cell line and to investigate the biological behavior of cisplatin-sensitive and cisplatin-resistant human ovarian cancer cells, and discusses the related mechanism.Then the related xenograft tumor models were established.Methods: Cell proliferation was assessed by soft agar growth test. Cell cycle and Cell apoptosis index was determined by flow cytometry. Capacity of invasion and migration of OVCAR-3 and OVCAR-3/CDDP cells in vitro and vivo were measured by Transwell Chamber. Xenograft tumor models were established in nude mice.Results: (1)OVCAR-3/CDDP cell lines were developed with stable resistance to cisplatin, the resistance index was 13.42;(2)The changes of cellular morphology were observed by phase-contrast microscope;(3)IC50(50% inhibiting concentration) and RI(resistance index) were detected by MTT assay;(4)As compared with the parent cells, OVCAR-3/CDD cells exhibited lower growth rate and the percentages of cells in G0/G1 phase were significantly increased while the percentage of S phase cells were decreased(P<0.05);(5)With the development of drug-resistance , the ability of invasion and migration of OVCAR-3/CDD cells was obviously enhanced;⑷In soft agar growth test, OVCAR-3 cells formed more cell clones than OVCAR-3/CDDP cells ((0.66%±0.09%) vs.(0.31%±0.07%),P<0.05).(6)As compared with the parent cells, OVCAR-3/CDDP cells had higher migration capability ((233.1±8.5) vs. (167.4±5.9), P<0.01)) and invasive capability ((143.6±9.1) vs. (95.8±6.2), P<0.01). (7)Apoptosis index of OVCAR-3/CDDP cells resulting from loss of cell-matrix interaction decreased comparing OVCAR-3 cells((7.78±1.32)% vs. (15.41±1.26)% , P<0.01). The xenotransplantion models of human cisplatin-resistant ovarian cancer in ten of ten (100%) nude mice were successfully constructed.Conclusion: There are difference in the ability of invasion, migration and colony formation rate between cisplatin-sensitive OVCAR-3 and cisplatin-resistant OVCAR-3/CDDP cells.Objective: To demonstrate the reversion effects of Triptorelin on the cisplatin resistance OVCAR-3 cells and to investigate the role of the EGFR-MAPK-ERK1/2 in reversion effects of Triptorelin on cisplatin resistance.Methods:⑴Cisplatin-resistant cell subline OVCAR-3/CDDP was induced by excessive concentration and intermittent action combined with low concentration and continuously exposure to cisplatin;⑵The doubling time (Td) and resistance index were detected by MTT assay;⑶The cell cycle and the cell apoptosis were determined by flow cytometry;⑷ERK1/2 activation, induced by cisplatin, Triptorelin and the combination (cisplatin and Triptorelin) different concentration, was determined by Western blot using an anti-phosphor-ERK antibody;⑸The Capacity of invasion and migration of OVCAR-3/CDDP cells treated with Triptorelin in vitro was measured by Transwell Chamber.⑹The EGFR expression of cells of every group was detected by flow cytometry.⑺The volume of subcutaneous tumors were measured.HE staining,the expression of EGFR was detected using immunohistochemistrical method.Results:⑴Continuous exposure of cell lines and xenografts to GnRH agonist resulted in growth inhibition of cancer cells in a dose- and time-dependent manner. In Cisplatin-resistant cell subline OVCAR-3/CDDP, the drug resistance reversal fold was 3.91. Active ERK1/2(p-ERK1/2) increased in the ovarian cancer cell subline OVCAR-3/CDDP;⑵A clear G0/G1 phase block in OVCAR-3/CDDP cells was shown after treatment with Triptorelin. Active ERK1/2(p-ERK1/2) in OVCAR-3/CDDP cells was up-regulated by cisplatin, but was down-regulated by either Triptorelin or combination.⑶GnRH agonists significantly reduced EGFR expression.⑷In vivo,the average volume of ovarian cancer in either Triptorelin or combination group was lower than those of control group(P<0.05).Conclusion:⑴Cisplatin-resistant cell subline OVCAR-3/CDDP was an essentially experimental model in vitro for the study on reversion effects of cisplatin resistance;⑵Triptorelin increased the cytotoxic effects of cisplatin in OVCAR-3/CDDP cells both in vitro and vivo;⑶I These effects were related to the regulation of the activity of the EGFR-MAPK-ERK1/2 signaling pathway.Objective: To investigate the inhibitory effect of gonadotropin-releasing hormone agonists on the migratory and the invasive behavior of cisplatin-resistant ovarian cancer cells and its molecular mechanism.Methods: Matrigel experiment was used to evaluated the effect of EGF,AG1478 or Triptorelin on invasive and migratory of OVCAR-3/CDDP cells. The protein Levels of MMP-2、TIMP-2、MMP-9 and TIMP-1 in cultured cell supernatants were determined by enzyme linked immunosorbent assay.The expression of P-EGFR protein in OVCAR-3/CDDP cells was determined by Western blot.Results: Matrigel experiment showed that EGF increased the invasion (P<0.01) and migration (P<0.01) ability of OVCAR-3/CDDP cells in vitro at the concentration of 10μg/L.AG1478 or Triptorelin inhibited EGF-induced invasion and migration of OVCAR-3/CDDP cells (P<0.01) by blocking the activity of EGFR. After treatmen with EGF the protein of P-EGFR was increased, which was abolished by AG1478. Enzyme linked immunosorbent assay revealed that exogenous EGF up-regulated protein levels of MMP-2 and MMP-9 and down-regulation protein levels of TIMP-1 and TIMP-2. Meanwhile AG1478 and Triptorelin reversed the effect of EGF by decreasing the levels of MMP-2,MMP-9 and increasing the levels of TIMP-1 and TIMP-2. The MMP-2 to TIMP-2 ratio and the MMP-9 to TIMP-1 ratio were decreased significantly by Triptorelin or AG1478 (P<0.01).Conclusion: Triptorelin can reduce the migratory and the invasive behavior of cisplatin-resistant ovarian cancer cells in vitro by changing MMP-2,MMP-9,TIMP-1 and TIMP-2 expression via EGFR signal pathway.更多还原