【作者】 田爽；

【导师】 辛晓燕； 李立文；

【作者基本信息】 第四军医大学 ， 妇产科学， 2009， 博士

【摘要】 卵巢癌是最常见的妇科恶性肿瘤之一,由于具有起病隐匿、早期不易发现、易转移、易发生铂类药物抵抗等特点,因此通常预后不佳。尽管近年来随着外科手术、化疗、放疗以及辅助治疗技术的不断发展,卵巢癌患者的5年生存率有所改善,然而,目前已有的治疗方案均不能达到完全治愈卵巢癌的目的。因此,研究影响卵巢癌迁移、耐药发生的分子机制,成为目前妇科肿瘤的研究重点与难点。近年来,流行病学研究和动物实验均表明慢性应激是影响肿瘤发生发展和演进的重要因素,尤其是交感神经递质去甲肾上腺素的作用逐渐受到重视。最初研究发现去甲肾上腺素可通过调节机体免疫系统功能促进恶性肿瘤的发生发展,如削弱自然杀伤(natural killer)细胞的细胞毒活性、抑制T细胞对有丝分裂原刺激的反应等。最近研究则发现去甲肾上腺素能够直接作用于肿瘤细胞,上调VEGF、bFGF、基质金属蛋白酶9(matrix metalloproteinase-9,MMP-9)等促血管生成分子表达,从而加速肿瘤组织生长和血管化,并且该作用可被β肾上腺素能受体拮抗剂心得安和β2肾上腺素能受体特异性小干扰RNA(small interfering RNA,siRNA)阻断,然而其分子机制尚未阐明。因为VEGF、bFGF和MMP-9的翻译起始过程均依赖于eIF4F复合物,ERK-Mnk1信号通路和Akt-mTOR信号通路可通过调节eIF4E磷酸化和4E-BP家族成员磷酸化影响eIF4F复合物活性,而去甲肾上腺素能够激活ERK-Mnk1信号通路和Akt-mTOR信号通路,所以我们推测去甲肾上腺素通过正性调控eIF4F复合物活性促进肿瘤增殖和血管化。同时,由于eIF4F复合物控制着survivin等在决定化疗敏感性中发挥关键作用的抗凋亡分子的翻译起始过程,所以我们推测去甲肾上腺素能够通过上调survivin表达诱发化疗抵抗。在此基础之上,我们尝试使用磷酸化缺陷型4E-BP2阻断eIF4F复合物装配,以期提高卵巢癌细胞的化疗敏感性,从而为建立新的卵巢癌靶向治疗策略奠定基础。目的:探讨去甲肾上腺素对卵巢癌细胞增殖、迁移及化疗敏感性的影响并初步阐明其分子机制,进而针对eIF4F复合物设计、制备新型蛋白治疗药物,评价其对卵巢癌化疗敏感性的影响。方法:①使用伤口愈合实验测定去甲肾上腺素对人卵巢癌SKOV3细胞迁移能力的影响;②应用MTT法和流式细胞术测定去甲肾上腺素对SKOV3细胞增殖能力的影响;③用Annexin-V/PI双标法和DAPI染色法测定去甲肾上腺素对SKOV3细胞顺铂敏感性的影响;④Western Blot法测定去甲肾上腺素对磷酸化Akt、磷酸化mTOR、磷酸化p70S6K、磷酸化4E-BP1、磷酸化ERK1、磷酸化Mnk1、磷酸化eIF4E、HIF-1α、c-myc、ODC、MDM2和survivin等表达水平的影响;⑤ELISA法测定去甲肾上腺素对VEGF分泌水平的影响;⑥设计、构建原核表达载体pVIP4 ,诱导表达并纯化融合蛋白PTD4-EGFP-t4EBP2;⑦MTT法绘制生长曲线观察PTD4-EGFP-t4EBP2对SKOV3细胞增殖的影响;⑧Annexin-V/PI双标法测定PTD4-EGFP-t4EBP2对SKOV3细胞顺铂敏感性的影响;⑨ELISA法测定PTD4-EGFP-t4EBP2对VEGF分泌水平的影响;⑩Western Blot测定PTD4-EGFP-t4EBP2对survivin表达水平的影响。结果:①去甲肾上腺素处理SKOV3细胞3h、6h和12h时划痕宽度分别为初始宽度的98.7±2.2%、95.4±3.0%和92.7±2.2%,而对照组则分别为93.0±2.4%、80.7±3.7%和69.8±2.6%其中6h和12h时与对照组相比差异非常显著(P<0.001)。②去甲肾上腺素对SKOV3细胞增殖的影响呈双相调节,在3h和6h时细胞增殖指数分别为39.6±1.3%和41.0±1.6%(P<0.001),9h时则为45.8±3.0%,而12h和24h进一步升高至70.4±3.8%和63.9±4.8%(P<0.001),MTT法绘制生长曲线表明细胞增殖能力明显增强。③使用去甲肾上腺素处理SKOV3细胞0h、3h、6h、9h、12h和24h后加入终浓度为10μg/mL顺铂,12h后Annexin-V/PI双标法测定细胞凋亡率显示在3h和6h时凋亡率明显升高(3h,P<0.001;6h,P<0.001),而在9h、12h和24h凋亡率则均显著下降(P<0.001),DAPI染色法结果与Annexin-V/PI双标法一致。④Western Blot显示去甲肾上腺素处理SKOV3细胞后磷酸化Akt、磷酸化mTOR、磷酸化p70S6K、磷酸化4E-BP1、磷酸化ERK1、磷酸化Mnk1、磷酸化eIF4E、HIF-1α、c-myc、ODC、MDM2和survivin等表达水平均明显升高;⑤DNA测序证实原核表达载体pET28a-PTD4和pVIP4与设计完全相符;⑥以SKOV3细胞总RNA为模板进行RT-PCR,成功获得了截短型4E-BP2 cDNA,克隆至pVIP4后经双酶切鉴定、PCR鉴定和DNA测序证实获得pVIP4-t4EBP2原核表达载体;⑦将pVIP4-t4EBP2转化至ER2566大肠杆菌后获得高效表达,经纯化制备了重组融合蛋白PTD4-EGFP-t4EBP2;⑧荧光显微镜观察显示融合蛋白PTD4-EGFP-t4EBP2能够高效进入SKOV3细胞,作用24h后VEGF分泌水平仅为对照组的21.0±2.7%(P<0.001),Western Blot显示其能显著抑制survivin表达;⑨在作用24h和48h时,融合蛋白PTD4-EGFP-t4EBP2对SKOV3细胞增殖的抑制率分别可达16.9±4.4%和47.8±6.2%,与使用PTD4-EGFP对照组相比差异显著(P<0.001);⑩使用重组蛋白PTD4-EGFP-t4EBP2干预SKOV3细胞24h即可使早期凋亡率上升至14.2±1.2%,与PTD4-EGFP组相比差异非常显著(P<0.001)。联合使用PTD4-EGFP-t4EBP2和顺铂可使早期凋亡率和晚期凋亡率分别达到45.2±3.3%和20.2±2.7%,与PTD4-EGFP+顺铂组相比差异非常显著(P<0.001)。结论:①去甲肾上腺素能够抑制SKOV3细胞迁移;②去甲肾上腺素对SKOV3细胞的顺铂敏感性呈双相调节,在早期提高顺铂敏感性,而在9h后显著诱发顺铂抵抗;③去甲肾上腺素能够上调c-myc、ODC、MDM2等增殖相关分子、抗凋亡分子survivin、促血管生成相关分子VEGF和HIF-1α表达,其机制可能涉及激活ERK-Mnk1信号通路促进eIF4E磷酸化和激活Akt-mTOR信号通路促进4E-BP1磷酸化;④成功制备了原核表达载体pVIP4-t4EBP2,在大肠杆菌ER2566中实现了PTD4-EGFP-t4EBP2的高效表达,并纯化获得了PTD4-EGFP-t4EBP2融合蛋白;⑤证实PTD4-EGFP-t4EBP2融合蛋白能够抑制SKOV3细胞增殖,下调VEGF的分泌水平和survivin的表达水平;⑥证实PTD4-EGFP-t4EBP2融合蛋白能够大幅度提高SKOV3细胞对顺铂的敏感性,提示eIF4F复合物是有效逆转化疗抵抗的关键靶点,并且阻断eIF4F复合物装配是提高化疗敏感性的有效手段。更多还原

【Abstract】 Ovarian cancer, the most common gynecologic malignancy, is the fifth leading cause of death from cancer in women and the leading cause of death from gynecological cancer. The evolution of surgical techniques, chemotherapy, radiotherapy and adjuvant therapy through multiple clinical trials over the past three decades has resulted in improvements in ovarian cancer treatment and increase in 5-year survival rate. However, the high incidence of platinum- resistance following first-line platinum-paclitaxel doublet treatment makes ovarian cancer incurable.Epidemiologic and experimental animal studies have shown that chronic stress promotes tumor development and progression and the sympathetic transmitter, noradrenaline, has been considered as an important mediator. Thus far, the majority of research on the deleterious effects of stress has focused on the neuroendocrine regulation of the immune response. In both animal and human studies, chronic stress has been shown to decrease cellular immune parameters, such as natural killer cell cytotoxicity and T-cell responses to mitogen stimulation. Recently, there is growing evidence confirming that alterations in neuroendocrine dynamics due to chronic stress can cause alterations in tumor pathogenesis.Several report demonstrated that noradrenaline could act on tumor cells directly and promote tumor growth and angiogenesis through up-regulation the expression of VEGF, bFGF and MMP-9. Further more, both propranolol and ADRB2 siRNA could completely block noradrenaline-induced effects. However, its molecular mechanisms had not been uncovered.As the translation initiation of VEGF, bFGF and MMP-9 depend on eIF4F complex, the ERK-Mnk1 pathway and Akt-mTOR pathway could regulate eIF4F activities by phosphorylation of eIF4E or 4E-BPs, and noradrenaline could activate both the ERK-Mnk1 pathway and Akt-mTOR pathway, we postulate that noradrenaline promote tumor growth and angiogenesis through positive regulation of eIF4F complex. Meanwhile, eIF4F complex controls the translation initiation of survivin, an anti-apoptotic protein which plays key roles in determination of chemosensitivity. Therefore, we suppose that noradrenaline could induce chemoresistance by up-regulation the translation of survivin. At last, we prepared a novel fusion protein composed of protein transduction domain and phosphorylation-defective truncated 4E-BP2, and investigated whether it could enhance the chemosensitivity of ovarian cancer cells by disruption of eIF4F assembly. Methods: Wound healing assay was carried out to detect the effect of norepinephrine on SKOV3 cell migration. Apoptosis rate and cell cycle phase distribution of SKOV3 cells were measured by flow cytometry (FCM). The effect of norepinephrine on cell apoptosis and chemosensitivity were examined by Annexin V/PI staining and DAPI staining. The expression levels of Akt、p-mTOR、p-p70S6K、p-4E-BP1、p-ERK1、p-Mnk1、p-eIF4E、HIF-1α、c-myc、ODC、MDM2 and survivin was analyzed by Western blot and the secretion of VEGF was examined by ELISA assay.After design and construction a prokaryotic expression vector pVIP4- t4EBP2, a novel fusion protein PTD4-EGFP-t4EBP2 was highly expressed in E.coli ER2566 and purified. The effect of PTD4-EGFP-t4EBP2 on SKOV3 cell proliferation was evaluated by MTT assay. Annexin-V/PI staining was used to evaluate the effect of PTD4-EGFP-t4EBP2 on cisplain sensitivity. The effects of PTD4-EGFP-t4EBP2 on VEGF secretion and survivin expression was examined by ELISA assay and Western blot, respectively.Results: After treatment of SKOV3 cells with noradrenaline, the mean remaining width at 3h, 6h and 12h was 98.7±2.2%, 95.4±3.0% and 92.7±2.2% of the original wound width, but the mean remaining width in the control group was 93.0±2.4%、80.7±3.7% and 69.8±2.6%. Noradrenaline decrease the proliferation index of SKOV3 cells to 39.6±1.3% and 41.0±1.6% at 3h and 6h (P<0.001), but at 12h and 24h, the proliferation index was increased to 70.4±3.8% and 63.9±4.8%(P<0.001). Cell growth curve demonstrated that the tumor cells proliferate very quickly between 12 and 24 hours. Annexin-V/PI staining demonstrated that at early phase, pretreatment SKOV3 cells with noradrenaline significantly promote cisplatin-induced apoptosis, but after 9 hours pretreatment, the tumor cells are resistant to cisplatin-induced death. Western Blot demonstrated that noradrenaline significantly increase the phosphorylation levels of ERK1, Mnk1 and eIF4E, suggested that ERK-Mnk1 pathway was activated. Meanwhile, phosphorylation levels of Akt, mTOR, p70S6K and 4E-BP1 were also elevated, suggested that Akt-mTOR pathway was another downstream signal pathway that was activated by noradrenaline. And HIF-1α, c-myc, ODC, MDM2 and survivin, whose translation initiation has been proved to depend on the activity of eIF4F complex were all up-regulated.DNA sequencing confirmed that prokaryotic expression vector pET28a- PTD4 and pVIP4 were successfully constructed. Truncated 4E-BP2 cDNA was amplified by RT-PCR using total RNA from SKOV3 cellls as template and subsequently cloned into pVIP4, double endonuclease cutting, PCR with specific primers and DNA sequencing suggested that pVIP4-t4EBP2 was successfully. After transforming pVIP4-t4EBP2 into E.coli ER2566 competent cells, recombinant protein PTD4-EGFP-t4EBP2 was highly expressed and prepared. Under fluorescence microscope, we find that fusion protein PTD4-EGFP-t4EBP2 could enter SKOV3 cells with high efficiency, and both the secretion of VEGF and the expression of survivin were down-regulated. After transduction of SKOV3 cells with PTD4-EGFP-t4EBP2 for 24 or 48 hours, the inhibition rate was 16.9±4.4% and 47.8±6.2%, respectively. And transduction of SKOV3 cells with PTD4-EGFP-t4EBP2 for 24 hours, the percent of early apoptotic cells of SKOV3 cells was elevated from 3.3±1.0% to 14.2±1.2%, and the percent of apoptotic cells induced by cisplatin was elevated from 16.3±1.8% to 45.2±3.3% (P<0.001).Conclusion: Though noradrenaline enhances the cisplatin sensitivity of SKOV3 cells at early phase, the tumor cells are resistant to apoptosis after 9 hours. Also, noradrenaline promotes the proliferation of SKOV3 cells after 9 hours, but an obvious inhibition could be detected at 3 hours or 6 hours. Moreover, noradrenaline could activate both ERK-Mnk1 pathway and Akt-mTOR pathway which were demonstrated by up-regulation the phosphorylation levels of ERK, Mnk1 and eIF4E, or the phosphorylation levels of Akt, mTOR, p70S6K and 4E-BP1. And noradrenaline could up-regulate the expression of c-myc, ODC, MDM2, survivin and HIF-1αand enhance VEGF secretion, all of which are involved in promoting tumor growth and angiogenesis, confirmed that eIF4F complex plays key roles in tumor progression. The recombinant fusion protein PTD4-EGFP-t4EBP2 significantly inhibits SKOV3 cell proliferation, VEGF secretion and sensitizes the tumor cells to cisplatin through survivin expression.更多还原