【作者】 李振宇；

【导师】 伍钢；

【作者基本信息】 华中科技大学 ， 肿瘤学， 2012， 博士

【摘要】 第一部分Auranofin对X线照射NSCLC细胞放射增敏的实验研究目的：体外观察Auranofin对肺腺癌细胞系A549的X线放射增敏效应。方法：采用MTT法检测Auranofin对人肺腺癌细胞A549和人肺大细胞癌细胞H460活性的影响,克隆形成法进行放射增敏实验,了解0、0.25μM、0.5μM和1.0μM的Auranofin预处理12h对A549细胞X线照射后存活分数的影响,通过多靶单击模型拟合成细胞存活曲线,了解各组细胞的放射敏感性参数。结果:Auranofin对A549和H460细胞活性的抑制呈浓度依赖性和时间依赖性。1.0μMAuranofin对A549有放射增敏的作用最为显著。克隆形成实验分析auranfin预处理的A549细胞照射后的存活分数,经多靶单击模型拟合成细胞存活曲线,结果0、0.25μM、0.5μM和1.0μM Auranofin预处理组放射抗拒参数SF2分别为0.827、0.718、0.653和0.393,1.0μM Auranofin对A549细胞的放射增敏比为3.0(DO值比)。结论：Auranofin可以增强NSCLC细胞的放射增敏性第二部分Auranofin对x线照射NSCLC细胞放射增敏的机制目的：研究Auranofin增加X线照射NSCLC细胞放射敏感性的机制。方法：分光光度计法和Western Bloting法分别检测终浓度为0、0.5和2.0μM的Auranofin对A549细胞TrxRl活性和HO-1的影响。Western Bloting法检测2.0μM的Auranofin预处理对x线照射4Gy后凋亡下游信号JNK/P38磷酸化、凋亡抑制家族IAP、BCL2、TRAF表达,Bax转位,PARP活化的影响。JC-1法检测2.0μM Auranofin对X线照射4Gy后A549细胞线粒体膜电位的变化。采用BIP抑制Bax的转移后,Auranofin对A549细胞放射敏感性的变化。结果：Auranofin可以明显的减少TrxRl活性和HO-1的表达。经Auranofin预处理,X线照射后JNK/P38的磷酸化更加明显,IAP、BCL2、TRAF家族蛋白表达减少,Bax向线粒体的转位增加,线粒体膜电位下降,活化的PARP片段增加。BIP阻断Bax向线粒体的转位后,翻转了Auranofin对A549细胞放射增敏作用。结论：Auranofin通过抑制TrxR活性和HO-1的表达,激活JNK/P38通路,抑制IAP、BCL2、TRAF表达,进而促进放射后Bax转位和细胞凋亡,增加非小细胞肺癌细胞的放射敏感性。第三部分Auranofin对肿瘤血管新生的影响目的：了解Auranofin对非小细胞肺癌的血管新生的影响。方法：Western biting检测0、0.5和2.0μM Auranofin对A549和H460细胞转录因子HIF1α, HIF2a和Spl,以及下游细胞因子VEGF单体和二聚体,VEGF165b单体和二聚体蛋白表达的影响。transwell小室法检测Auranofin对与A549细胞共培养的HUVECs侵袭能力的影响。结果：Auranofin对A549细胞HIF1α的影响不明显,但增加H460细胞HIFla的表达。Auranofin可以减少A549和H460细胞HIF2α和Sp1的表达,VEGF单体和二聚体的表达,增加抑制性VEGF165b单体和二聚体的表达。Auranofin明显抑制与A549细胞共培养的HUVECs侵袭能力。结论：Auranofin影响转录因子的表达和VEGF的表达,抑制非小细胞肺癌的血管新生。更多还原

【Abstract】 PartⅠAuranofin Enhances Radiation sensitivity of NSCLC cellsObjective:Our goals in this study were to determine the role of Auranofin on radiation sensitivity of human non-small cell lung cancer cells in vitro.Methods:MTT assays were performed to study the effect of Auranofin on cell viability of human adenocarcinoma cells A549and human large cell lung cancer cells H460. We further analyzed cell survival of a combined treatment which A549cells underwent0,0.25,0.5or1.0μM Auranofin pretreatment firstly and irradiated with different doses of X-ray radiation afterwards, and calculated the parameters of radiation sensitivity.Result:Auranofin inhibited the cell viability of A549and H460cells on dose-dependent and time-dependent manner. The SF2of cell survival culve of0,0.25,0.5and1.0μM Auranofin pretreatment radiation group is0.827、0.7、0.653and0.393, respectively. The SER of1.0μM Auranofin pretreatment A549cells is3.0.Conclusion:Auranofin could enhance the radiation sensitivity of NSCLC cells. Part IIThe mechanism of Auranofin on radiation sensitivity in non small cell lung cancerObjective:Our goals in this study were to research the mechanism Auranofin enhance the radiation sensitivity of non small cell lung cancerMethods:Modified DTNB reduction assay was performed to determine TrxR activity of A549cells treated with Auranofin. HO-1protein was analysis by Western bloting after Auranofin treatment. phospho-JNK/P38, protein expression of anti-apoptosis family IAP, BCL2, and TRAF, mitochondrial potential, translocation of Bax to mitochondrial and cleaved PARP was determined when A549cells were treated by Auranofin pretreatment followed by radiotherapy.Results:By using the specific inhibitors Auranofin, activity of TrxR1and expression of HO-1protein were reduced. In Auranofin-radiotherapy treatment A549and H460cells, increasing of phospho-JNK/P38, protein expression inhibition of antiapoptosis family IAP, Bcl-2, and TRAF, mitochondrial potential decreasing, translocation of Bax to mitochondrial and cleaved PARP after X-ray radiation were developed to a much higher extent than X-ray radiation or Auranofin treatment only. By blocking Bax translocation using Bax inhibiting peptide, the Auranofin-enhanced radiosensitivity of A549was reduced.Conclusion:Our data suggest that Auranofin reduced the activity of TrxR and expression of HO-1protein, induced translocation to mitochondrial of bax, triggerd the apoptosis of cells exposes to radiation, and finally enhanced the radiation sensitivity of NSCLC cells. Part ⅢThe effect of Auranofin on angiogensis of lung cancerObjective:The present studies were designed to test the hypothesis that Auranofin negatively regulates VEGF through effects on transcriptional factor HIF1α, HIF2a or Sp1expression.Methods:To test this hypothesis, we first examined the levels of HIF1α, HIF2a or Sp1in A549and H460cells treatment with Auranofin. Then, we studied the effects of Auranofin on VEGF by Western. To overview the total effect of data above on angiogensis. Tranwells assay was perform to value the analysis of HUVECs when co-culture with A549cells treated with AuranofinResults:In Auranofin-treated A549and H460cells, decreasing of HIF2α, Spl, VEGF and increasing VEGF165b was observed. But Auranofin did not alter HIF1α expression in A549cells and increasing in H460cells. Auranofin enhanced the invasion of HUVECs when co-culture with A549cells.Conclusion:Auranofin could alter Transcriptional factor HIF1α, HIF2α and Sp1and VEGF expression in NSCLC cells and contribute to the inhibition of invasion of HUVECs.更多还原