【作者】 邹伟；

【导师】 辛晓燕； 杨红；

【作者基本信息】 第四军医大学 ， 妇产科学， 2008， 博士

【摘要】 卵巢癌是严重威胁广大妇女生命的常见恶性疾病,其发病率有逐年上升的趋势,死亡率高居女性生殖系统恶性肿瘤之首。本病起病隐匿,早期诊断困难,一经发现多为晚期,目前尚无预防卵巢癌的有效方法。尽管已广泛开展肿瘤细胞减灭术以及术后联合铂类为基础的化疗的方案,但卵巢癌患者的5年存活率仍徘徊在25-30%[1],85%-90%的患者最终仍难免复发和转移,其重要原因是化疗耐药以及化疗毒副反应大,患者难以承受[2; 3]。因此,探讨肿瘤细胞化疗耐药机制和开发新型辅助化疗药物以增强铂类药的化疗效果、减少其毒副作用成为近年来妇科肿瘤学者研究的热点。环氧化酶(COX)是花生四烯酸生物合成前列腺素的限速酶,有COX-1和COX-2两种同工酶。其中COX-2(环氧化酶-2)的过度表达参与了肿瘤的发生和生长[4-8],同时参与多种肿瘤的侵袭和转移[9-11],而且与肿瘤的化疗耐药相关[12; 13]。研究发现COX-2抑制剂能够抑制多种实体肿瘤的生长、侵袭和转移,而且可增加多种化疗药物对肿瘤的细胞毒性[14-18]。已有研究发现在卵巢癌中存在COX-2的稳定表达,而在正常组织中无表达[19; 20]。目前仅有少量关于COX-2抑制剂单独应用于卵巢癌的研究报道,但结果存在争议,机制不明[21-23],塞莱昔布作为COX-2的选择性抑制剂,其与化疗药物联合作用于卵巢癌细胞的研究尚未见报道。研究目的本实验主要研究塞来昔布对卵巢癌细胞SKOV3生长、侵袭和对顺铂药物敏感性的影响,并探讨其可能的分子机制。研究方法第一部分塞来昔布对卵巢癌细胞SKOV3生长的抑制作用及作用途径研究本实验通过Proliferation Assay、流式细胞仪细胞周期分析、流式细胞仪细胞凋亡检测等方法研究塞来昔布对卵巢癌细胞SKOV3的增殖、凋亡的影响,同时与siRNA介导的COX-2沉默对SKOV3细胞的上述影响进行比较,探讨COX-2在其中是否起作用,并通过Western blot技术检测相关蛋白(Caspase-9, Caspase-3, PARP, Bcl-2, Bax, Cyclin D1)的变化情况。第二部分塞来昔布对卵巢癌细胞SKOV3对顺铂化疗敏感性的增强作用及作用途径研究本研究通过Proliferation Assay法检测低浓度(10μM,临床可达到的血药浓度)塞莱昔布对卵巢癌SKOV3细胞顺铂敏感性的增强作用,同时以特异性沉默COX-2表达的SKOV3/COX-2i细胞作为COX-2的阴性对照组细胞,探讨COX-2在其中是否起作用,并通过Western blot技术检测相关蛋白(Caspase-9, Caspase-3, PARP, Bcl-2, Bax, P-gp)的变化情况。第三部分塞来昔布对卵巢癌细胞SKOV3侵袭能力的抑制作用及作用途径研究本研究通过细胞基质粘附实验检测各组细胞间的粘附能力,利用transwell小室实验及划痕实验检测各组细胞的侵袭和迁移能力,同时与siRNA介导的COX-2沉默对SKOV3的引起的上述影响进行比较,明胶酶谱法(Zymography)检测MMP-2、MMP-9的活性,Western blot技术检测MMP-2、MMP-9、E-cadherin蛋白的变化情况。研究结果1. Proliferation Assay结果显示:24h、48h、72h时,高浓度Celecoxib(50μM)和特异性沉默COX-2表达后可明显抑制卵巢癌细胞SKOV3生长,而低浓度(2μM,10μM)效果不明显;接着流式细胞仪检测细胞周期和细胞凋亡情况,细胞周期结果显示:50μM Celecoxib作用24h和特异性沉默COX-2表达的SKOV3/COX-2i,两组细胞G0/G1期细胞比例增加,S期及G2/M期下降,G1峰之前出现典型的亚二倍体峰;流式细胞仪凋亡检测结果显示:凋亡早期细胞簇群,Annexin V(+)PI(-)和凋亡晚期细胞或死亡细胞簇群,Annexin V(+)PI(+)的细胞明显高于空白对照组和10μM Celecoxib组; Western blot结果显示50μM Celecoxib作用24h和特异性沉默COX-2表达后,SKOV3细胞的细胞周期蛋白Cyclin D1表达量减少,而与凋亡执行密切相关的Caspase-3、Caspas-9和PARP均出现了活化的片段,而Bcl-2家族的Bcl-2和Bax表达无明显变化。10μM Celecoxib作用24h后上述蛋白无相应变化。2.与正常对照组细胞相比,10μM Celecoxib与10μM Cisplatin联合作用SKOV3细胞24h后能明显抑制SKOV3细胞的生长(p<0.05),而单独用10μM Cisplatin未见明显的生长抑制作用(p>0.05)。两者联合作用于COX-2表达阴性的SKOV3/COX-2细胞后,观察到类似的生长抑制效果。Western blot结果显示联合用药组细胞出现Caspase-9、Caspase-3和PARP的活化片段,Bcl-2、Bax表达无明显变化,而单独用10μM Cisplatin后上述蛋白无明显变化;正常SKOV3细胞几乎不表达多药耐药基因MDR1编码的P-gp蛋白,经10μM Cisplatin作用24h后,P-gp蛋白表达增加,而10μM Celecoxib与10μM Cisplatin联合用药能抑制由Cisplatin引起的P-gp表达增加。3.细胞基质粘附实验结果显示:10μM Celecoxib作用24h后,粘附于Matrigel胶上的SKOV3细胞数明显多于未经Celecoxib处理的SKOV3细胞(p<0.05);侵袭实验结果显示:经10μM Celecoxib作用24h后,SKOV3细胞穿过人工基底膜的细胞数明显少于未经Celecoxib处理的SKOV3细胞(p<0.05);划痕实验结果提示:经10μM Celecoxib作用24h后,SKOV3细胞迁移至损伤区的细胞数明显少于未经Celecoxib处理的SKOV3细胞(p<0.05);转染COX-2 siRNA的SKOV3/COX-2i在上述3个实验中较之正常对照组细胞组无明显差异(p>0.05)。凝胶酶谱实验和Western blot结果显示:经10μM Celecoxib作用24h后,SKOV3细胞MMP-2、MMP-9的活性和表达量受到明显抑制;Western blot结果显示E-cadherin的表达经Celecoxib作用24h后,细胞较之正常对照组和特异性沉默COX-2的SKOV3/COX-2i,E-cadherin表达明显增强。结论1.高浓度的Celecoxib(50μM)可以通过抑制细胞周期蛋白Cyclin D1的表达,活化“Caspase/PARP”通路,实现抑制SKOV3细胞增殖和促进细胞凋亡的作用;通过RNAi技术特异性沉默SKOV3细胞COX-2的表达也可通过上述途径抑制SKOV3细胞的增殖和促进细胞凋亡,但作用要弱于50μM Celecoxib;该作用途径存在COX-2依赖和COX-2非依赖途径,而整个过程是Bcl-2非依赖的。2.小剂量Celecoxib(10μM)与Cisplatin联合用药可抑制SKOV3细胞中由Cisplatin引起的多药耐药基因MDR1编码的P-gp表达增加,活化“Caspase/PARP”通路,增强SKOV3对化疗药Cisplatin的敏感性;通过RNAi技术特异性沉默SKOV3细胞COX-2的表达不能增加SKOV3细胞对Cisplatin的敏感性;小剂量Celecoxib(10μM)对SKOV3细胞对Cisplatin的增敏作用是COX-2非依赖的。3.小剂量Celecoxib(10μM)通过增加E-cadherin的表达,抑制MMP-2、MMP-9的表达和活性,增强SKOV3细胞的粘附力,抑制其侵袭和迁移力;通过RNAi技术特异性沉默SKOV3细胞COX-2的表达不能引起上述变化;小剂量Celecoxib对SKOV3细胞侵袭的抑制作用是COX-2非依赖的。4.Celecoxib具有良好的抗肿瘤作用,其有效浓度10μM是临床作为非甾体抗炎药应用时可达到的,其有望成为新的化疗辅助药,具有很好的临床应用前景。更多还原

【Abstract】 Ovarian cancer is one of the most common life-threatening malignant tumors among women and it is the first leading cause of death from gynecologic cancers. Due to the lack of effective screening strategies and the absence of symptoms in early-stage of disease, majority of cases have progressed to an advanced stage at the time of primary diagnosis. Although most patients will undergo an attempt at surgical debulking followed by 6 cycles of chemotherapy with a platinum-based regimen, the 5-year survival remains 25-30%[1] and 85-90% patients will develope tumor recurrence or metastasis. Cisplatin as the critical component in the chemotherapy regimens, its usage has been limited because of its cumulative toxicities, especially the toxicity to the kidney and the intrinsic or acquired resistance to cisplatin in some patients[2; 3]. In recent years, the interests of combining cisplatin and new active chemotherapeutic agents to treat ovarian cancer have arisen for promoting the effectiveness and reducing the side effect of cisplatin.Cyclooxygenase (COX) is an important speed-limited enzyme in the conversion of arachidonic acid to prostaglandins. Two isoenzymes of COX, COX-1 and COX-2, have been idendified. Overexpression of COX-2 is known to be closely associated with tumor growth[4-8], metastasis[9-11] and chemotherapy resistance[12; 13] in several kinds of human tumors. The selective inhibitor of COX-2 has been shown to suppress tumor growth, invastion and metastasis in solid tumors, and increase the cytotoxicity of chemotherapy drugs to tumor cells[14-18]. It has been found that COX-2 is stably expressed in ovarian cancer tissue, but not in normal tissues[19; 20]. Although there are several reports about COX-2 inhibitors used in ovarian cancer, the results are debatable and the mechanism is unclear. There is no report about the combination of celecoxib with chemotherapy drug in ovarian cancer cells nowaday.Objective1. To investigate the effect and mechanism of COX-2 selective inhibitor Celecoxib on cell proliferation in human ovarian cancer cell line SKOV3.2. To investigate the effect and mechanism of Celecoxib on cell sensitivity to Cisplatin in human ovarian cancer cell line SKOV3.3. To investigate the effect and mechanism of Celecoxib on cell ivasion in human ovarian cancer cell line SKOV3.MethodsPart One the effect and mechanism of COX-2 selective inhibitor Celecoxib on cell proliferation in human ovarian cancer cell line SKOV3The proliferation assay and flow cytometry were carried out to investigate the effect of Celecoxib on cell proliferation and apoptosis in ovarian cancer cell line SKOV3. As a comparison, the same work was done in SKOV3 cells in which the COX-2 expression was inhibited by COX-2 siRNA. Western blot was carried out to assess the changes of the expression of related proteins (Caspase-9, Caspase-3, PARP, Bcl-2, Bax, Cyclin D1).Part Two the effect and mechanism of COX-2 selective inhibitor Celecoxib on cell sensitivity to Cisplatin in human ovarian cancer cell line SKOV3.The proliferation assay was carried out to assess the enhancement effect of 10μM Celecoxib (clinical available) on cell sensitivity to Cisplain. The SKOV3/COX-2i cell, in which the COX-2 protein expression was inhibited by COX-2 siRNA, was used as the COX-2 negtive control to observe whether the COX-2 expression is dependent in the enhancement effect of Celecoxib on cell sensitivity to Cisplain. Western blot was carried out to assess the changes of the expression of related proteins (Caspase-9, Caspase-3, PARP, Bcl-2, Bax, P-gp).Part Three the effect and mechanism of COX-2 selective inhibitor Celecoxib on cell invasion in human ovarian cancer cell line SKOV3.The cell adhesion assay, transwell chamber and scratch wound assay were carried out to detect the abilities of adhesion, invasion and migration in SKOV3. The activity of MMP-2 and MMP-9 were examined by Zymography, the protein expression of COX-2, MMP-2, MMP-9 and E-cadherin was analyzed by Western blot. The same work was done in SKOV3/COX-2i to investigate the potential role of COX-2 in the changes caused by Celecoxib.Reults1. The cell growth was inhibited obviously in SKOV3 cells with high concentration of Celecoxib(50μM) at 24h, 48h and 72h, or in SKOV3/COX-2i in which the COX-2 protein expression was inhibited by COX-2 siRNA, but not in SKOV3 cells with low concentration of Celecoxib(2μM,10μM). Compared with the control and SKOV3 with Celecoxib 10μM , the SKOV3 with 50μM Celecoxib at 24h and SKOV3/COX-2i were retarded in G0/G1 phase and percentage of them in G0/G1 phase increased significantly and G1 subpeak before G1 phase peak(apoptosis peak) was shown. Compared with the control and SKOV3 with 10μM Celecoxib, the early apoptosis cells ( Annexin V(+)PI(-) ) and late apoptosis cells ( Annexin V(+)PI(+) ) were increased significantly. Western blot results showed that the expression of cell cycle protein Cyclin D1 was decreased and the amount of the active subunit of caspase-9 , caspase-3 and the cleaved subunit of PARP was increased, however, neither Bcl-2 nor Bax. Compared with the control, no significant changes of above-mentioned proteins were observed in SKOV3 with 10μM Celecoxib.2. Our data indicated that a remarkable increase of growth inhibition was observed in SKOV3 with the combination of 10μM Celecoxib with 10μM Cisplatin(p<0.05), nevertheless the treatment with 10μM Celecoxib or 10μM Cisplatin alone had little effect to induce growth inhibition(p>0.05). The same result was shown in SKOV3/COX-2i in which the COX-2 expression was inhibited specifically by COX-2 siRNA. In combination treatment groups, Western blot results showed that the amount of the active subunit of caspase-9 , caspase-3 and the cleaved subunit of PARP was increased, however, neither Bcl-2 nor Bax. Compared with the control, no significant changes of above-mentioned proteins were observed in SKOV3 with 10μM Cisplatin. Western blot results showed that MDR1 gene coded P-gp protein was hardly expressed in SKOV3 and SKOV3 with the combination of 10μM Celecoxib with 10μM Cisplatin for 24h, but increased in SKOV3 with 10μM Cisplatin for 24h.3. Treated with 10μM Celecoxib for 24 hours, cell adhesive assay, transwell chamber assay and scratch wound assay indicated that the adhesive, invasive and migratory ability of SKOV3 cells was inhibited significantly(P<0.05). Zymography results showed the descended activity of MMP-2 and MMP-9 and Western blot results showed the unchanged protein expression of COX-2 and decreased protein expression of MMP-2, MMP-9 and E-cadherin in SKOV3 cells treated with 10μM Celecoxib for 24 hours. But the same changes were not observed in SKOV3/COX-2i .Conclusions1. High concentration of COX-2 selective inhibitor Celecoxib (50μM) could inhibit the cell proliferation and increase the cell apoptosis of human ovarian cancer cell SKOV3 by inhibiting the cell cycle protein Cyclin D1 and activating the "Caspase/PARP" pathway. Specifical inhibition of COX-2 by COX-2 siRNA could inhibit the cell proliferation and increase the cell apoptosis of SKOV3, but the effect was no as good as Celecoxib (50μM). The effect mentioned above of Celecoxib might be through both COX-2 dependent and independent way, but Bcl-2 independent way.2. The combination of low concentration of Celecoxib (10μM) with Cisplatin (10μM) could enhance the sensitivity of SKOV3 to Cisplatin by inhibiting the expression of MDR1 gene coded P-gp increased by Cisplatin and activating the "Caspase/PARP" pathway. But the sensitivity of SKOV3 to Cisplatin could not be enhanced by specifical inhibition of COX-2 by COX-2 siRNA. The enhancement of sensitivity of SKOV3 to Cisplatin by low concentration of Celecoxib (10μM) might be COX-2 independent.3. Low concentration of Celecoxib (10μM) could increase the adhension and inhibit invasion and migration of SKOV3 by increasing the expression of E-cadherin and inhibiting the expression and activation of MMP-2 and MMP-9. Specifical inhibition of COX-2 by COX-2 siRNA could not cause the changes mentioned above. The inhibition of invasion of SKOV3 caused by low Celecoxib might be COX-2 independent.4. Celecoxib has a positive antitumor effect and the effective concentration of 10μM is clinical available when it is used as NSAIDs. It might be a new chemotherapy adjunctive drug and has a potential clinical prospect.更多还原