【作者】 蔡国青；

【导师】 辛晓燕； 王建；

【作者基本信息】 第四军医大学 ， 妇产科学， 2007， 博士

【摘要】 卵巢癌的耐药性问题是其临床治疗效果欠佳的主要原因之一。明确卵巢癌耐药的分子机制,寻找有效的耐药逆转剂或有效的治疗靶标已成为国内外妇科肿瘤学界密切关注的研究热点。糖原合成激酶3β(glycogen synthesis kinase-3β,GSK-3β)是一多功能丝氨酸/苏氨酸类激酶,广泛的分布在迄今研究过的所有真核生物中,是糖原代谢过程中的重要限速酶,可以在胰岛素调控下磷酸化肝糖原合成酶(glycogen synthesis)使之失活。业已证实,除此功能之外,GSK-3β在多个重要信号通路中扮演了关键角色,例如PI3’-Kinase、Wnt/wingless以及Hedgehog三条信号转导通路,在蛋白合成、细胞增殖、细胞分化、细胞运动以及肿瘤发生等方面都扮演了重要角色。已有研究报道,相对于正常卵巢组织,卵巢癌中存在GSK-3β的高表达。2006年,Qi Cao等证明:来源于卵巢上皮性癌的细胞系SKOV3中GSK-3β活性高于来源于卵巢透明细胞癌的细胞系ES-2;利用LiCl抑制SKOV3中GSK-3β活性会抑制SKOV3细胞增殖能力,认为其可能成为新的卵巢癌治疗靶标。但按照Qi Cao等的结论逆推,ES-2细胞中GSK-3β活性较低,理应增殖能力较低,恶性程度也应较低。可事实并非如此,临床实践证明,卵巢透明细胞癌虽然早期患者居多,但其易复发,化疗耐药性发生率高于其它卵巢肿瘤。且已有报道,抑制GSK-3β活性会增加一些肿瘤细胞的耐药性。这些证据令我们对抑制GSK-3β活性治疗卵巢癌的可能性提出质疑。经过文献回顾,有关GSK-3β与卵巢癌耐药机制之间的研究尚未见报道,因此,我们决定对GSK-3β活性与卵巢癌耐药性之间的联系进行研究,以进一步探讨抑制GSK-3β活性治疗卵巢癌的可能性。研究目的:1.明确GSK-3β表达及活性改变是否参与了卵巢癌耐药性的发生; 2.探讨外源性改变GSK-3β活性或表达对卵巢癌细胞耐药性的影响。研究方法:1. western bllot、免疫细胞化学染色检测A2780、CP70细胞中GSK-3β蛋白及其磷酸化蛋白的表达、定位情况;western bllot检测顺铂处理后A2780、CP70细胞中GSK-3β蛋白表达及定位的改变。2. LiCl抑制GSK-3β活性后,MTT法检测A2780、CP70细胞IC50的改变。3. DAPI染色、Annexin V/PI染色流式细胞仪检测LiCl抑制GSK-3β活性对顺铂诱导A2780、CP70细胞凋亡的影响。4.利用pcDNA -GSK-3β-S9A质粒瞬时转染CP70细胞,观察GSK-3β活性增强对CP70细胞耐药性的影响。5.利用特异性GSK-3βsiRNA瞬时转染CP70细胞,观察GSK-3β表达及活性受到抑制后,CP70细胞耐药性的改变。6.改变CP70细胞内GSK-3β活性后,利用HGA700原子吸收光谱仪测定顺铂处理后细胞中DNA-铂结合量,观察其DNA修复能力的改变。7.改变CP70细胞内GSK-3β活性后,westernblot检测GSK-3β下游相关分子的表达变化情况,初步探讨CP70细胞内GSK-3β活性改变导致DNA修复能力变化的分子机制。研究结果:1.细胞总蛋白提取物中,A2780、CP70细胞中总的GSK-3β蛋白表达无明显差异(1:1.08±0.13,P>0.05);而CP70细胞中,处于活性抑制状态的pGSK-3β-ser9表达量明显高于A2780细胞(1:2.34±0.21,P<0.01)。2. A2780细胞中,GSK-3β主要定位于胞浆中,胞核中几乎没有;而CP70细胞中,GSK-3β胞浆、胞核中均有表达;使用相应浓度顺铂处理A2780、CP70细胞后,无论A2780细胞还是CP70细胞,顺铂处理均会导致胞浆中GSK-3β移位至胞核中。3.未用顺铂处理时,A2780、CP70细胞胞浆中GSK-3β表达及活性状态未见明显差异;顺铂处理后,两种细胞胞核内均出现GSK-3β移位,且pGSK-3β-ser9表达量增加明显。4. LiCl预处理A2780、CP70细胞6小时可明显增加其顺铂耐药性(IC50值如下:A2780细胞,0.51±0.13μM:3.04±0.28μM,P<0.001;CP70细胞,5.43±0.15μM:13.28±0.34μM,P<0.001)。5. DAPI染色及Annexin V/PI染色证明LiCl预处理A2780、CP70细胞6小时可明显降低顺铂诱导的细胞凋亡发生。6. pcDNA -GSK-3β-S9A质粒瞬时转染CP70细胞可明显增加GSK-3β的表达,但GSK-3βser9的磷酸化过程受到阻断,致使CP70细胞耐药性降低(2.78±0.09μM:5.38±0.12μM,P<0.01)。7.利用特异性GSK-3βsiRNA瞬时转染CP70细胞可明显抑制GSK-3β蛋白的表达,致使CP70细胞耐药性增加(9.36±0.14μM:5.38±0.12μM,P<0.01)。8. GSK-3β活性受到抑制后,CP70细胞DNA修复功能明显增强;而GSK-3β活性增强后,CP70细胞DNA修复功能减弱。9. GSK-3β活性受到抑制后,p53、p21、GADD45等蛋白表达增强是CP70细胞DNA修复功能增强的原因之一,细胞免于顺铂诱导的凋亡;cleaved caspase-3表达受到抑制可能也参与其中。研究结论:1.胞核内pGSK-3β-ser9表达增强,是CP70细胞区别于A2780细胞的生物学特征之一。2.顺铂刺激可以引起A2780、CP70细胞中,GSK-3β蛋白的核移位及胞核内pGSK-3β-ser9表达的增强。3. LiCl预处理A2780、CP70细胞可以增强两种细胞的耐药性。4. LiCl引起的细胞耐药性增强与LiCl引起细胞内pGSK-3β-ser9表达增强有关。5.在CP70细胞中过度表达GSK-3βS9A突变体蛋白可以降低CP70细胞耐药性。6. RNAi技术沉默CP70细胞中GSK-3β蛋白的表达可以增强CP70细胞耐药性。7. p53蛋白及其下游产物p21、GADD45表达增加介导了GSK-3β活性受抑引起的肿瘤细胞耐药性改变;caspase-3激酶失活可能也参与其中。综上所述,卵巢癌耐药机制十分复杂,GSK-3β活性受到抑制后引起p53蛋白及其下游产物p21、GADD45表达增加是卵巢癌耐药性产生的可能机制之一。虽然有证据表明,抑制GSK-3β活性可抑制肿瘤细胞增殖能力,但鉴于抑制GSK-3β活性会导致肿瘤细胞耐药性增加,GSK-3β抑制剂能否作为卵巢癌治疗用药仍值得商榷。更多还原

【Abstract】 Ovarian carcinoma is a leading cause of death from gynecological cancer in China and many other countries. The major reason for the mortality of this cancer is the high metastatic ability and the resistance to chemotherapy. The response of the primary tumor to taxane and platinum-based chemotherapy is high, but about 20% of patients never achieve a clinical response and the majority of the patients will relapse and eventually die of drug-resistant disease. Although great improvement has been made in standard therapy for ovarian cancer, the mechanism of the drug-resistant and relapses is not clear yet. Identifying the molecules that differentially expresses in the drug-resistant cells is a hot field for exploring the target for new drugs.Glycogen synthase kinase-3 beta (GSK-3β), a serine/threonine kinase active in resting cells, is now recognized as a key component of a surprisingly large number of cellular processes, such as cellular structure, growth ,motility and apoptosis. Dysregulation of the expression and activity of GSK-3βhas been linked to several prevalent pathological conditions, including tumorigenesis.It has been reported that the level of expression of GSK-3βis significantly high in ovarian carcinoma tissue.Because of the importance of GSK-3βin apoptotic procedure, it is plausible that the pharmacological inhibiton of GSK-3βshould cure ovarian carcinoma. And the latest report also indicated that GSK-3βpositively regulates the proliferation of ovarian carcinoma cell line SKOV3, via the upregulation of cyclin D1, which can be inhibited by lithium chloride, an approved GSK-3βinhibitor(12). However, in this context, we offered the contrary evidence that the inhibiton of GSK-3βby lithium chloride offered the chemoresistance of ovarian carcinoma cells to cisplatin, and the molecular mechanisms responsible for the chemoresistance focused on the induction of p53, p21 and GADD45.AIM:Detect the role of GSK-3βin the cisplatin chemoresistance of ovarian cancer cells.METHODS:Our research work was divided into two parts. The first, we detected total protein and its phosphoralation isform expression of GSK-3βin A2780 and CP70 cells by western blot and immunofluorescence. Then we detect the change of cisplatin chemoresistance of A2780 and CP70 cells pretreated with LiCl by MTT, DAPI staining and flow cytometry.The second, after transient transfection of S9A mutant GSK-3βvector and GSK-3βsiRNA in CP70 cells, the change of cisplatin chemoresistance and the ability of DNA repairment were detected by MTT and DAPI staining, flow cytometry and atomic absorption spectroscopy. And then, the possible molecular mechanism was detected by western blot.At last, the data was analyzed with SPSS 13.0 statistical software.RESULTS:First, we detected the expression and localization of total GSK-3βand its inhibitive phosphorylated isoforms, pGSK-3βser9 in A2780 and CP70 cells. Western blot analysis showed that both A2780 and CP70 expressed GSK-3β, and the levels of total GSK-3βin two cell lines were almost aequalis (1:1.08±0.13,P>0.05), however, the level of pGSK-3βser9 isoforms in A2780 was lower than in CP70 (1:2.34±0.21,P<0.01). As we know, the function of GSK-3βis closely correlated with its localization. So the localization of GSK-3βwas detected by immunofluorescence. We found that GSK-3βalmost localized in cytoplasmic in A2780, but in cytoplasmic and nucleus in CP70. After treatment of cisplatin, GSK-3βtranslocated obviously into the nucleus of the two cell lines ,and pGSK-3βser9 is the main composition of GSK-3βin the nucleus.To confirm whether the increase of pGSK-3βser9 contributes to the cisplatin-resistant of A2780, CP70 cell lines, we examined the inhibitory concentration 50% (IC50) of cisplatin to A2780 and CP70 pretreated with lithium chloride. IC50 of A2780 and A2780/CP70 respectively significantly increased from 0.51±0.13μm, 5.43±0.15μm to 3.04±0.28μm , 13.28±0.34μm after lithium pretreatment. The results of DAPI and flow cytometry confirmed these consequence.To further verify that the effect of lithium was due to inhibition of GSK-3β, CP70 cells were transiently transfected with pcDNA3-GSK-3β-S9A and GSK-3βshort interfering RNA (siRNA), respectively. We observed that overexpression of GSK-3β-S9A resulted in the depression of cisplatin chemoresistance in CP70 cell, on the contrary, inhibition of GSK-3βexpression by siRNA led to the increase of cisplatin chemoresistance.It has been demonstrated that the cytotoxicity of cisplatin is thought to be due to the formation of intrastrand and interstrand cross-links in the DNA and cisplatin-DNA adducts, which may induce cell cycle arrest or apoptosis. So platinum cotent was measured by atomic absorption spectroscopy to determine the ability of removal of platinum-DNA adducts. The results of atomic absorption spectroscopy indicated that overexpression of GSK-3β-S9A resulted in the reduction of DNA repairment ability.As we know, in A2780 and CP70 cells, the resistance to cisplatin correlates with prolonged p53 protein stabilization and accumulation. Therefor, we examined the expression of p53 protein in CP70 cells, in which GSK-3βS9A mutant overexpressed or GSK-3βexpression was inhibited by siRNA. The results demonstrated that the inhibition of GSK-3βexpression resulted in an obvious increase of p53 expression, compared with those transfected with pcDNA3-GSK-3β-S9A, and this increase was associated with up-regulation of p21 and GADD45, two target genes of p53, which were correlated with cell cycle arrest and DNA repair. Furthermore, we found the expression of cleaved caspase-3 was reduced when p53 was induced by the inhibition of GSK-3β.CONCLUSIONS:Our results shows high level expression and nuclear location of pGSK-3βser9 in CP70 cells is associated with its resistance to cisplatin and lithium is able to promote the chemoresistance to cisplatin through induction of inactive phosphoralation of GSK-3βat serine 9 and p53. Therefore, the administration of lithium to ovarian cancer patients should be scrupulous.更多还原