【作者】 张海燕；

【导师】 孙红；

【作者基本信息】 复旦大学 ， 妇产科学， 2009， 博士

【摘要】 卵巢癌(Ovarian cancer,OC)是常见的女性生殖系统恶性肿瘤之一,严重威胁妇女健康和生活质量。上皮性卵巢癌是最常见的卵巢癌病理类型,占卵巢癌的85%～90%,其中75%的上皮性卵巢癌属于浆液性囊腺癌。2008年美国公立癌症监督机构(National Cancer Institute’s Surveillance,Epidemiology and EndResults,SEER)的统计报告显示,卵巢癌在全球范围年发生率大约13.3/100,000女性,亚洲每年发生率大约9.8/100,000女性。由于缺乏有效的筛查手段,大约67%的患者确诊时已发生腹腔或远处转移,错过手术的最佳时机,预后较差。卵巢癌的复发、转移以及对化疗抵抗成为其治疗的瓶颈。PI3K/AKT信号通路是细胞代谢和抗凋亡的重要转导途径。研究表明,该通路不仅与多种恶性肿瘤的发生、发展和预后相关,并且与多种一线化疗药物关系密切。本课题组的前期研究发现,PI3K分子在上皮性卵巢癌组织中过度激活,其抑制剂LY294002可增强顺铂对卵巢癌SKOV3细胞的杀伤作用。在此基础上,本课题拟进一步检测PI3K-p85α及其下游AKT在正常卵巢组织-良性卵巢浆液性囊腺瘤-交界性卵巢浆液性囊腺瘤-卵巢浆液性囊腺癌组织中的表达以及与临床病理特征的相关性;利用RNA干扰技术和真核表达质粒双向调节卵巢浆液性囊腺癌SKOV3细胞中AKT的表达,并运用RT-PCR、western blot、CCK-8、Cloneformation、FCM、Wound healing、Transwell-Matrigel等方法检测其在细胞增殖、凋亡、运动和侵袭中的作用及机制。同时,采用顺铂作用于上皮性卵巢癌SKOV3、ES-2细胞,western blot检测不同时间、不同浓度梯度的顺铂刺激对细胞中AKT/mTOR磷酸化水平的影响情况;建立单层培养的耐药细胞株SKOV3/DDP和肿瘤细胞团簇SKOV3/MCA;western blot检测两种耐药模型中AKT/mTOR信号通路的激活情况;利用RNA干扰技术抑制SKOV3和SKOV3/DDP细胞中AKT的表达,并运用MTT和western blot探讨其在顺铂耐药中的作用及机制。最后,采用抑制剂Triciribine和Rapamycin,联合顺铂作用于SKOV3和ES-2细胞,运用MTT和FCM比较不同治疗方案的疗效,为卵巢癌治疗提供实验基础。本课题分为以下四个部分。第一部分PI3K-p85α、AKT在卵巢浆液性囊腺癌中的表达及临床意义目的检测PI3K-p85α、AKT在正常卵巢组织-良性卵巢浆液性囊腺瘤-交界性卵巢浆液性囊腺瘤-卵巢浆液性囊腺癌组织中的表达,探讨其与卵巢浆液性囊腺癌的临床病理特征的相关性。方法收集正常卵巢组织20例,良性卵巢浆液性囊腺瘤组织15例,交界性卵巢浆液性囊腺瘤组织15例和卵巢浆液性囊腺癌组织24例。运用RT-PCR和westernblot分别检测组织中PI3K-p85α、AKT在mRNA和蛋白水平的表达,并分析其与临床病理特征的相关性。结果(1) PI3K-p85αmRNA在正常卵巢组织-良性卵巢浆液性囊腺瘤-交界性卵巢浆液性囊腺瘤-卵巢浆液性囊腺癌组织中均有表达,但卵巢浆液性囊腺癌组织中的表达水平(1.008±0.223)明显高于正常组、良性组以及交界性组(分别为0.167±0.092,0.389±0.095和0.576±0.037)(均为P＜0.05);(2) PI3K-p85α蛋白在正常卵巢组织中未检测到表达,但83%卵巢浆液性囊腺癌组织可检测其阳性表达;(3) PI3K-p85αmRNA水平、蛋白阳性表达率与上皮性卵巢癌的临床分期和组织分级相关(均为P＜0.05),但与患者年龄无关(P＞0.05);(4) p-AKT是AKT的活性形式。p-AKT在正常卵巢组织-良性卵巢浆液性囊腺瘤-交界性卵巢浆液性囊腺瘤-卵巢浆液性囊腺癌组织中均有表达,但卵巢浆液性囊腺癌组织的表达水平明显高于正常组、良性组以及交界性组(均为P＜0.05);(5)AKT1、AKT2和AKT3 mRNA在正常卵巢组织-良性卵巢浆液性囊腺瘤-交界性卵巢浆液性囊腺瘤-卵巢浆液性囊腺癌组织中组织中均有表达,但卵巢浆液性囊腺癌组织的表达明显高于正常组、良性组以及交界性组(均为P＜0.05);(6)p-AKT表达水平、总AKT mRNA水平与上皮性卵巢癌的临床分期和组织分级相关(均为P＜0.05),但与患者年龄(均为P＞0.05)。结论PI3K-p85α、AKT在卵巢浆液性囊腺癌组织中异常激活,并且与临床病理分期和组织学分级相关联。AKT异常激活不仅是上游PI3K分子激活的后续效应,同时与AKT自身扩增密切相关。第二部分AKT激酶对卵巢浆液性囊腺癌细胞增殖、凋亡和侵袭的作用及机制目的探讨AKT激酶活性的改变对卵巢浆液性囊腺癌SKOV3细胞增殖、凋亡、运动和侵袭的影响和机制。方法应用重组IGF-1作用于SKOV3细胞,western blot检测细胞中AKT的磷酸化情况。针对AKT1基因,设计并构建shRNA质粒和真核表达质粒,双向调节SKOV3细胞中AKT1的表达,运用RT-PCR和western blot检测转染效率。运用CCK-8、Clone formation、FCM、Wound healing、Transwell-Matrigel等方法检测转染前后细胞增殖、凋亡、运动和侵袭的变化。RT-PCR法检测与细胞运动侵袭相关分子CXCR4、VEGF、MMP-2、MMP-9和uPA在mRNA水平的表达变化。结果(1)IGF-1迅速诱导SKOV3细胞中AKT磷酸化,激活AKT通路,作用45min达到高峰,随后呈下降趋势并维持在一定水平。(2)成功构建AKT1基因的真核表达质粒pEF-1α-AKT1和靶向抑制AKT1基因的shRNA表达质粒pRNAT-AKT1。转染SKOV3细胞后,能有效调控p-AKT表达。(3)参照未转染组和空载体转染组,外源性AKT1可显著促进细胞增殖,促进细胞侵袭和运动,抑制细胞凋亡,促使进入G2-S期的细胞增多(均为P＜0.05);外源性AKT1可明显上调CXCR4、VEGF、MMP-2和uPA的mRNA表达,但对MMP-9 mRNA的影响不明显。(4)shRNA靶向抑制AKT1基因的表达可显著抑制细胞增殖,抑制细胞侵袭和迁移,促进细胞凋亡,导致细胞周期G0-G1期阻滞(均为P＜0.05);抑制AKT1表达可明显下调CXCR4、VEGF、MMP-2和uPA的mRNA表达,但对MMP-9 mRNA的影响不明显。结论AKT影响着卵巢浆液性囊腺癌SKOV3细胞的增殖和凋亡,并可能通过调控CXCR4、VEGF、MMP-2和uPA的转录水平来影响细胞运动和侵袭能力。第三部分AKT/mTOR信号通路在上皮性卵巢癌顺铂化疗中的作用及机制目的检测顺铂对上皮性卵巢癌细胞中AKT/mTOR信号通路的影响以及AKT/mTOR信号通路在耐药模型中的激活情况,探讨AKT/mTOR信号通路在顺铂化疗耐药中的作用及机制。方法Western blot检测不同时间或浓度梯度顺铂刺激对上皮性卵巢癌SKOV3、ES-2细胞中AKT/mTOR磷酸化水平的影响。采用低剂量递增法诱导单层培养的耐药细胞株SKOV3/DDP,MTT法检测其耐药指数。采用液相重叠系统建立三维培养的肿瘤细胞团簇。台盼蓝细胞计数验证SKOV3/DDP和SKOV3/MCA对顺铂化疗的抵抗性。Western blot检测AKT/mTOR信号通路在两种耐药模型中的激活情况。利用shRNA靶向抑制SKOV3和SKOV3/DDP细胞中AKT1的表达,MTT检测SKOV3和SKOV3/DDP细胞对顺铂化疗的敏感性变化,western blot检测与细胞生存相关的survivin和与耐药相关的P-gp蛋白表达水平的变化。结果(1)在一定浓度时间范围内,顺铂刺激可产生类IGF-1样作用,诱导AKT、mTOR激活。在作用时间上,顺铂20μmol/L作用5min,SKOV3、ES-2细胞中p-AKT、p-mTOR表达增加,30min左右最显著,随后至120min略呈下降趋势;在作用浓度上,顺铂20μmol/L作用120min,p-AKT、p-mTOR表达增加,随着顺铂浓度的增加略呈下降趋势。(2)诱导获得耐药细胞株SKOV3/DDP,耐药指数约为4.62。建立三维陪养的肿瘤细胞团簇SKOV3/MCA,台盼蓝细胞计数检测证实其对顺铂存在抵抗性(P＜0.05)。(3)参照SKOV3细胞,AKT/mTOR信号通路在SKOV3/DDP细胞和SKOV3/MCA中异常激活(P＜0.05)。(4)AKT siRNA不仅显著抑制SKOV3细胞和SKOV3/DDP细胞的生长,并削弱了SKOV3/DDP细胞对顺铂的抵抗性(P＜0.05)。(5)AKT siRNA可下调survivin蛋白和P-gp蛋白在SKOV3/DDP和SKOV3细胞中的表达。结论在一定浓度时间范围内,顺铂刺激可诱导上皮性卵巢癌细胞中AKT、mTOR激活;耐药模型中AKT、mTOR呈异常激活状态,提示AKT/mTOR信号通路异常可能是耐药机制之一;AKT siRNA可增强SKOV3细胞对顺铂的敏感性,削弱SKOV3/DDP细胞对顺铂的抵抗性,其机制可能是通过下调survivin蛋白和P-gp蛋白的表达。第四部分Triciribine、Rapamycin增强上皮性卵巢癌细胞对顺铂的敏感性目的比较单独Triciribine/Rapamycin、单独顺铂或联合两种制剂作用对上皮性卵巢癌SKOV3、ES-2细胞增殖和凋亡的影响,为上皮性卵巢癌联合化疗提供实验依据。方法采用western blot检测Triciribine/Rapamycin对AKT/mTOR的抑制效应,筛选治疗浓度。采用单独Triciribine/Rapamycin、单独顺铂或联合两种制剂作用于上皮性卵巢癌SKOV3和ES-2细胞,运用MTT和FCM比较不同治疗方案对其增殖和凋亡的影响。结果(1)在正常培养条件下,10μmol/L Triciribine作用24h,上皮性卵巢癌细胞内p-AKT表达显著抑制,同时抑制细胞生长;参照单独顺铂作用和单独Triciribine作用,联合两种制剂作用显著增加SKOV3和ES-2细胞的凋亡率(均为P＜0.05)。(2)在正常培养条件下,100nmol/L Rapamycin作用24h,上皮性卵巢癌细胞内p-mTOR表达显著抑制,同时抑制细胞生长;参照单独顺铂作用和单独Rapamycin作用,联合两种制剂作用显著增加SKOV3和ES-2细胞的凋亡率(均为P＜0.05)。结论Triciribine、Rapamycin分别通过抑制AKT、mTOR激活而增强顺铂的敏感性;顺铂联合抑制剂为上皮性卵巢癌的临床治疗提供了一种值得尝试的治疗方案。更多还原

【Abstract】 Epithelial ovarian cancer is the leading killer among gynecological malignancies. About 67% of patients in epithelial ovarian cancer initially present with advanced stage, missing good chance for operation. Then chemotherapy becomes one of the important treatments. Unfortunately, parallel to high efficacy in the treatment, the development of resistance is a major obstacle. Previous studies indicated that the PI3K/AKT pathway played a pivotal role in many human malignances, including ovarian cancer. Increased activation of AKT was noted in ovarian cancer, which exerted anti-apoptotic effect, antagonized cell cycle arrest, modulated angiogenesis, and mediated mRNA translation through mTOR signaling. However, a limited amount of information was available regarding the function of the PI3K/AKT pathway in the context of cisplatin-induced apoptosis in ovarian cancer.This study was scheduled to analyze the expression of PI3K/AKT in epithelial ovarian cancer and examine the involvement of the PI3K/AKT pathway in human ovarian cancer cell line SKOV3 in the forms of monolayer and three-dimensional cell cultures, as models of tumor cells in peritoneal dissemination of ovarian cancer. Furthermore, comparison between parental and drug-resistant cells would be taken to explore important clues for successful cisplatin therapy. The identification of the PI3K/AKT signaling responsible for cisplatin-based chemotherapy may provide more targets for combination therapy aimed to circumvent drug-resistance.Part I Expression and clinicopathologic meaning of PI3K-p85α、AKT in serous epithelial ovarian carcinomaObjective To explore the expression and significance of phosphatidylinositol-3 kinase-p85αand AKT at protein and mRNA levels in serous epithelial ovarian carcinoma.Methods The expression of PI3K-p85αand AKT at protein and mRNA levels were evaluated by western blot and RT-PCR in normal ovarian epithelium(group N, n=20), benign serous ovarian tumors(group Bl , n=15), borderline serous ovarian tumors(group B2, n=15) and serous epithelial ovarian carcinoma(group E, n=24).The relevant clinical pathological parameters were analyzed. Results The tendency of two methods was coincidence on the whole. There was no positive expression of PI3K p85αprotein in group N, but 13% in group B2, 83% in group E. PI3K-p85αmRNA was 0.167±0.092 in group N, 0.389±0.095 in group B1, 0.576±0.037 in group B2 and 1.008±0.223 in group E. There was significant difference between group N, group B1, group B2, and group E(P<0.05). There was significant difference between p85αexpression and tumor differentiation degree and clinicalopathological staging (P <0.05, P <0.05). Overactivation of AKT was detected in group E, compared with group N, group B1, and group B2(P<0.05). Three isoforms of AKT mRNA were detected in each group. However, AKT1, AKT2, and AKT3 mRNA were overexpressed in group E compared with group N, group B1, and group B2(P<0.05). Significant differences were noted between AKT activation and tumor differentiation degree and clinicopathological staging (P <0.05, P <0.05).Conclusions Overactivation of PI3K/AKT signaling existed in serous epithelial ovarian carcinoma. The alteration of PI3K-p85αand AKT were correlated with clinicopathological staging and tumor differentiation degree.Part II Effects of AKT activation on cell proliferation, apoptosis, migration, and invasion in epithelial ovarian cancer cellsObjective To explore the effects of activation of AKT on cell growth, cell cycle, cell invasive, cell migration, cell clone formation and cell apoptosis, and to explore related mechanism.Methods Western blot was used to detecte the activation of AKT by IGF-1 stimulation. Recombinant plasmids carrying the full-length AKT1 cDNA or AKT1 siRNA were constructed. Plasmids were transfected into SKOV3 cells. Western blot and RT-PCR were used to detect the transfection efficiency. Flow cyctometry was used to detect cell early apoptosis rate and cell cycle progression. CCK-8 and clone formation assay were used to examine cell proliferation. Transwell-Matrigel assay was used to analyse cell invasion. Wound healing assay was used to analyse cell migration. RT-PCR was used to explore the expression of several cell migration related moleculars at mRNA level.Results Western blot detected the intact activation of AKT in SKOV3 cells by IGF-1 stimulation on time dependent manner. A plasmid (pEF-1α-AKT1) containing full length of AKT1 cDNA and specific AKT1-targeted shRNA expression plasmids were constructed successfully, which could effectively upregulate or downregulate the activation of AKT after transfected into SKOV3 cells. Compared with untranfected cells or non-target shRNA transfected cells, down-regulation AKT1 inhibited cell growth and clone formation, decreased cell migration and invasion, and reduced cell population in the S phase(P <0.05), but induced cell apoptosis. Additionally, down-regulation AKT1 decreased the expression of CXCR4, VEGF, MMP-2, and uPA at mRNA level(P<0.05). On the other hand, up-regulation AKT1 increased cell migration, proliferation, and invasion(P<0.05). Up-regulation AKT1 increased the expression of CXCR4, VEGF, MMP-2, and uPA at mRNA level(P <0.05).Conclusions AKT played an important role in cell growth and suvivial in epithelial ovarian cancer, and might impact cell migration and invasion by regulating the expression of CXCR4, VEGF, MMP-2, and uPA at mRNA level.Part III Role and mechanism of AKT/mTOR signaling in cisplatin-based chemotherapy in epithelial ovarian cancer cellsObjective To investigate the role and mechanism of AKT/mTOR signaling in cisplatin-based chemotherapy in epithelial ovarian cancer cells.Methods Cisplatin was administrated at different concerntrations and different times. Western blot was used to examine the activation of AKT and mTOR in SKOV3 and ES-2 cells. SKOV3/DDP monolayer cells and SKOV3/MCA (multicellular aggregates) were constructed as chemoresistant models. Western blot was used to detect the expression of PI3K/AKT signaling in both models comparing with SKOV3 cells. The role and mechanism of AKT siRNA in different models before and after cisplatin treatment were detected by MTT assay. Western blot was used to check the expression of survivin and multidrug resistant protein before or after AKT siRNA transfection.Results Cisplatin increased the activation of AKT and mTOR dramatically on dose-dependent and time-dependent manners in SKOV3 and ES-2 cells. SKOV3/DDP monolayer cells and SKOV3/MCA were constructed and proved as cisplatin-resistant models. Elevated activation of AKT/mTOR signaling was observed in SKOV3/DDP cells and SKOV3/MCA(P<0.05). AKT siRNA could attenuate cisplatin resistance dominantly(P<0.05) and decreased the protein expression of survivin and P-gp.Conclusions Cisplatin could play an anti-apoptotic role by inducing the activation of AKT and mTOR in epithelial ovarian cancer cells. The AKT/mTOR/survivin signaling was involved in cisplatin-based chemoresistance in epithelial ovarian cancer. AKT siRNA could enhaced the effects of cisplatin-based chemotherapy by decreasing the expression of survivin and P-gp.Part IV Enhanced effects of Triciribine or Rapamycin on cisplatin-based chemotherapy in epithelial ovarian cancer cellsObjective To explore the effects of Triciribine or Rapamycin singly or combined with cisplatin on cell proliferation and apoptosis in SKOV3 and ES-2 cells.Methods Western blot detected the optical concerntration of Ticiribine and Rapamycin in cells. Methyl thiazolyl tetrazolium and flow cytometry were used to detect the growth rate and apoptosis rate after cisplatin-based chemotherapy, Triciribine or Rapamycin treatment or combination treatment, respectively.Results (1) The activation of AKT was dramatically inhibited after exposured to 10μmol/L Triciribine under normal culture condition(P <0.05). Triciribine enhanced the sensitivity of cisplatin-based chemotherapy in SKOV3 and ES-2 cells. (2) The activation of mTOR was dramatically inhibited after exposured to 100nmol/L Rapamycin under normal culture condition(P <0.05). Rapamycin enhanced the sensitivity of cisplatin-based chemotherapy in SKOV3 and ES-2 cells.Conclusions Both Triciribine and Rapamycin could enhanced cisplatin-based chemotherapy in epithelial ovarian cancer cells by inhibiting corresponding taget.更多还原