【作者】 王佳瑞；

【导师】 王琪；

【作者基本信息】 大连医科大学 ， 内科学， 2010， 博士

【摘要】 研究背景及目的：肺癌是常见的恶性肿瘤之一,其发病率和致死率都居各种肿瘤之首。化疗耐药是影响肺癌疗效的主要因素之一。肺癌的耐药主要分为原发性耐药和继发性耐药,前者表现为肿瘤细胞对化疗药物的天然不敏感,后者是肿瘤细胞因抗癌药物诱导或其他因素的激活而产生的耐药性。肺癌对化疗药物的耐药是一个多因素参与的复杂过程,主要与化疗药物的药代动力学、肿瘤细胞特异性、肿瘤细胞生存的微环境三大因素密切相关。而且各种因素之间相互依赖、相互制约、相互调节。化疗药物种类繁多,目前常用于肺癌临床治疗的主要有VP-16、顺铂等。VP-16属于鬼臼乙叉甙类,是细胞周期特异性药物。以顺铂为代表的铂类药物是临床治疗各种类型肺癌的常规用药,通过形成链内或者链间交联影响DNA的复制、合成,引起细胞的凋亡。细胞特异性因素与细胞内一系列耐药相关蛋白的表达及其介导的耐药密切相关。目前研究较多的与肺癌有关的耐药相关蛋白主要有P-糖蛋白(P-glycoprotein,P-gp),多药耐药相关蛋白(multidrug resistance-associated protein,MRP),肺耐药相关蛋白(lung resistance-related protein,LRP),谷胱甘肽-S-转移酶-π(Glutathione-s-transferase-π,GST-π)和拓扑异构酶Ⅱα(TopoisomeraseⅡα, TopoⅡα).研究表明P-gp、MRP、LRP.GST-π和TopoⅡα在肺癌的原发性和继发性耐药中都起着重要的作用。但是国内外的相关研究大多集中于其中的一种或几种,将五种耐药相关蛋白联合检测以及关于它们与肺癌耐药相关性的报道甚少。除了细胞特异性因素可以引起化疗耐药之外,耐药还与肿瘤生存的微环境密切相关。由于血供不足,实体瘤通常处于一种低氧、低糖、酸中毒的微环境中。这种特殊的微环境通常会导致肿瘤细胞的葡萄糖调节应激反应,诱导糖调节蛋白(glucose-regulated proteins,GRPs)的表达。目前研究最多的是糖调节蛋白78(GRP78)和94(GRP94)。我们以前通过分别对四种细胞系进行研究发现,肺癌细胞中GRP78和GRP94的表达与VP-16的耐药有关,但不同细胞系之间的差异以及机制尚不清楚。众所周知,肺癌化疗耐药是一个复杂的过程,并非单一因素,单一蛋白作用的结果。耐药相关蛋白和糖调节蛋白可能共同参与了肺癌对化疗药物的耐药,但两者之间的关系尚不清楚,深入研究两组蛋白的相互关系以及在肺癌耐药中的作用,有利于提高化疗疗效。为了全面的了解多种耐药相关蛋白在肺癌化疗耐药中的作用、糖调节蛋白对不同肺癌细胞系耐药的影响和机制以及耐药相关蛋白与糖调节蛋白之间的关系,本课题开展以下研究。研究内容：1.耐药相关蛋白在肺癌原发性耐药中的作用采用RT-PCR、Western blot和免疫荧光的方法对耐药相关蛋白P-gp、MRP、LRP、GST-π和TOPOⅡα在四种不同类型肺癌细胞系(SK-MES-1、SPCA-1、NCI-H-460和NCI-H-446)中进行定性定量检测,并用MTT法检测各细胞系对顺铂、阿霉素和VP-16的耐药性。分析耐药相关蛋白在肺癌对顺铂、阿霉素和VP-16耐药中的作用。2.耐药相关蛋白在肺癌继发性耐药中的作用采用RT-PCR、Western blot和免疫荧光的方法对五种耐药相关蛋白在肺腺癌顺铂耐药细胞株A549/DDP和亲代细胞株A549的表达进行检测,分析耐药相关蛋白在肺腺癌顺铂继发性耐药中的作用。3.P-gp的下调对肺癌耐药的影响采用RT-PCR和免疫荧光的方法检测不同类型肺癌细胞系维拉帕米处理前后P-gp的表达,MTT法检测维拉帕米处理前后不同类型肺癌细胞系对顺铂和VP-16耐药性。分析P-gp在不同细胞系对顺铂和VP-16耐药中的影响。4.糖调节蛋白对肺癌耐药的影响采用RT-PCR、Western blot和免疫荧光的方法检测A23187作用下四种不同类型肺癌细胞系GRP78和GRP94的表达以及不同细胞系对VP-16的耐药性,并在此基础上,进一步探讨GRP78和GRP94的表达与肺癌对VP-16耐药的关系。5耐药相关蛋白和糖调节蛋白在肺癌耐药中的相互作用研究采用RT-PCR、Western blot和免疫荧光的方法检测耐药相关蛋白和糖调节蛋白在四种肺癌细胞系的基础表达。分析两组蛋白之间的相关性和对肺癌化疗耐药的影响。研究结果：1.耐药相关蛋白对肺癌原发性耐药的影响RT-PCR.Western blot和免疫荧光的实验结果表明：P-gp.MRP.LRP.GST-π和TOPOⅡα五种耐药相关蛋白的蛋白及mRNA水平在四种不同类型肺癌细胞系中皆有表达,但表达水平有差异。比如GST-π在肺鳞癌细胞系SK-MES-1表达水平最高,但是TOPOⅡα在SK-MES-1表达水平却最低。MTT结果显示：四种不同类型细胞系对阿霉素、顺铂及VP-16的化疗敏感性不同。肺腺癌SPCA-1细胞系对阿霉素和顺铂最敏感,但是肺鳞癌SK-MES-1对VP-16最敏感。GST-π和TOPOⅡα与肺癌对阿霉素、顺铂及VP-16的原发性耐药有相关性。GST-π蛋白水平的表达与肺癌对顺铂耐药成正相关(P<0.05),TOPOⅡα蛋白水平的表达与肺癌对VP-16的耐药成正相关(P<0.05),但其表达与肺癌对阿霉素的耐药成负相关(P<0.05)。2.耐药相关蛋白对肺癌继发性耐药的影响RT-PCR.Western blot和免疫荧光的实验结果表明：与亲代细胞株相比,肺腺癌顺铂耐药株A549/DDP中P-gp和MRP表达显著增高(P<0.05),TOPOⅡα表达明显降低(P<0.05)。3.P-gp的下调对肺癌耐药的影响RT-PCR.免疫荧光的实验结果显示：维拉帕米能够明显抑制四种肺癌细胞系P-gp核酸和蛋白水平的表达(P<0.05)。MTT结果表明：P-gp表达水平的下调增加了小细胞肺癌NCI-H-446对顺铂的敏感性(P<0.05),也增加了肺腺癌SPCA-1,大细胞NCI-H-460及小细胞肺癌NCI-H-446对VP-16的敏感性(P<0.05)4.糖调节蛋白对肺癌原发性耐药的影响RT-PCR-Western blot和免疫荧光的实验结果表明：GRP78和GRP94在四种细胞系中都有表达,但是各细胞系GRP78基础表达在蛋白和核酸水平均存在差异(P<0.05,在NCI-H-460细胞系中表达最高);而GRP94没有显著性差异。在诱导剂A23187作用下,四种细胞系中GRP78蛋白和核酸水平的表达显著增加(P<0.05),而GRP94仅在蛋白水平显著增加。MTT结果显示：在A23187作用下,GRP78和GRP94高表达组的细胞对VP-16的IC50值明显高于低表达组(P<0.05)。GRP78蛋白和核酸基础表达与肺癌对VP-16耐药成正相关(P<0.05)。在A23187作用下,四种细胞系中GRP78和GRP94蛋白水平表达都与VP-16耐药成正相关(P<0.05)：GRP78核酸水平表达与SK-MES-1.SPCA-1和NCI-H-446细胞系对VP-16的耐药明显相关(P<0.05) GRP94核酸水平表达与四种细胞系对VP-16无明显相关。5.耐药相关蛋白和糖调节蛋白在肺癌耐药中的相互作用RT-PCR.Western blot和免疫荧光的实验结果表明：耐药相关蛋白P-gp.MRP.LRP.GST-π和TopoⅡα与糖调节蛋白GRP78和GRP94在四种不同类型肺癌细胞系中均有表达,并且GRP78和GRP94的表达与除P-gp以外的耐药相关蛋白表达趋势基本一致。GRP78与LRP蛋白水平的表达成正相关(r=0.773,P<0.05)。二者协同参与肺癌的化疗耐药。结论：1.P-gp.MRP.LRP.GST-π和TOPOⅡα五种耐药相关蛋白在四种肺癌细胞系中的共表达可能是肺癌对化疗药物产生原发性耐药的主要原因之一。细胞间耐药相关蛋白表达的差异性可能是造成其化疗疗效不同的重要因素之一。其中,GST-π蛋白水平的表达与肺癌对顺铂原发性耐药明显相关,TOPOβα蛋白水平的表达与肺癌对阿霉素的化疗敏感性相关同时也与肺癌对VP-16原发性耐药相关。2.P-gp.MRP和TOPOⅡα的表达与肺腺癌对顺铂继发性耐药相关,三种耐药相关蛋白可能共同参与了肺腺癌对顺铂的继发性耐药。3.维拉帕米能够显著抑制四种细胞系P-gp核酸和蛋白水平的表达,P-gp表达水平的下调与小细胞肺癌对顺铂耐药相关,与肺腺癌、大细胞癌和小细胞癌对VP-16耐药相关。4.GRP78的基础表达与肺癌对VP-16的耐药成正相关,细胞间GRP78表达的差异可能是不同类型肺癌对VP-16化疗敏感性差异的原因之一。GRP78和GRP94蛋白水平的表达与四种肺癌细胞系对VP-16的耐药都有显著相关性,GRP78核酸水平的表达与SK-MES-1.SPCA-1和NCI-H-446三种细胞系对VP-16耐药相关。5.耐药相关蛋白和糖调节蛋白之间具有一定的相互影响,它们共同在肺癌的耐药中起着重要作用。联合检测耐药相关蛋白和糖调节蛋白及探讨两者之间的关系一方面揭示肺癌耐药的机制,判断化疗疗效和预后,另一方面可以根据肺癌耐药相关蛋白的表达合理选择化疗药物,正确配伍,减少化疗药物引起的副作用,还可以根据其产生耐药的机制选择相应的逆转剂,有利于提高临床化疗疗效。更多还原

【Abstract】 Background and Aim:As one of the most frequent malignancies, Lung cancer is the leading cause of cancer death in the world. Drug resistance remains a major problem for successful chemotherapy in lung cancer. Chemoresistance can be primary(intrinsic) where tumor cells do not respond to anticancer drugs from the beginning of the treatment or secondary (acquired) where tumor cells develop resistance during chemotherapy. As is well known, Drug resistance is a complex process mainly involving with pharmacokinetics, cancer-cell-specific issues and the tumor microenvironment. Various factors are mutually dependent and inter-acting in development of dug resistance。There are varieties of anticancer drugs for the treatment of lung cancer, such as VP-16, cisplatin. VP-16, one member of epiopodophyllotoxin, is the cell cycle specific drugs. Platium characterized by cisplatin can be used to treat all types of lung cancer. Through formation of cross-link, cisplatin affect the replication and synthesis of DNA and cause cell apoptosis.Cancer-cell-specific issues can be mediated by changes of a series of intracellular drug resistance-related protein. Among which, P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), Glutathione-s-transferase-π(GST-π) and Topoiso-meraseⅡα(TopoⅡα) has drew more attention. Many reports showed that P-gp, MRP, LRP, GST-π, and TopoⅡαplayed important roles in the intrinsic or acquired resistance in lung cancer. However, most of them only focused on one or two of these drug resistance related proteins, little information is known referring to all of these five mediators.In addition to cancer-cell-specific factors, mocroenviroment may also a crucial factor influencing chemoresistance in lung cancer. Because of poor vascularization, solid tumors usually contain hypoxic regions where glucolysis is the main means of ATP synthesis, and glucose deprivation often occurs, which trigger the glucose-regulated stress response of cancer cells, leading to the dramatically increased expression of glucose-regulated proteins(GRPs). GRP78 and GRP94 are the best studied GRPs. Our previous research showed that A23187 can induced the expression of GRP78 in multiple lung cancer cell lines, and its expression is related to the resistance to VP-16. Meanwhile, A23187 induced GRP94 expression is associated with the resistance to VP-16 as well in lung cancer. However, these results are all about the sigle cell line. In this work, we investigated the expression of GRP78 and GRP94 and evaluate whether their different expression can result in resistance discrepancy to VP-16 in mutiple lung cancer cell lines.Mutiple factors are suggested to be involved in drug resistance in lung cancer. Both drug resistance related proteins and GRPs can be important related contibutors. However, little is known about the correlationship between these factors.This work aimed at clarifying the effect of several drug resisitance related factors in lung cancer including P-gp, MRP, LRP, GST-πand TopoIIa on intrinsic and acquired drug resistance, the relationship between expression of GRPs and chemoresistance. Further more, correlation among them was well analyzed according to comprehensive factors mentioned above.Methods:1. The expression of P-gp, MRP, LRP, GST-πand TopoⅡwas examed by RT-PCR, Western blot and/or immunofluorescence in four histopa-thological subtypes of lung cancer cell lines (SK-MES-1, SPCA-1, NCI-H-460 and NCI-H-446) and the resistance to cisplatin, doxorubicin and VP-16 was detected by MTT assay. Analysis the relationship between the drug resistance-related protein and the resistance to cispaltin, doxorubicin and VP-16 in lung cancer.2. The expression of the drug resistance-related proteins in cisplatin- resistance lung adenocarcinoma cell line A549/DDP and the parental cell line A549 was detected by RT-PCR, Western blot or immunofluorescence3. The expression of P-gp with and without the pretreatment of verapamil was examed by RT-PCR and immunofluorescence in four histopa-thological subtypes of lung cancer cell lines. Cell survival to cisplatin and VP-16 was determined by MTT assay.4. The expression of GRP78 and GRP94 pretreated with A23187 was detected by RT-PCR, Western blot and immunofluorescence. MTT assay was used to determined cell survival to VP-16.5.Pearson correlation analysis was emplored to determine the relation-ship between drug resistance related proteins and GRPs so as to supply benefit indicator for individual treatment of lung cancer.Results:1. We found that the endogenous levels of P-gp, MRP, LRP, GST-πand TOPOⅡαin four cell lines vary. The level of GST-πin SK-MES-1was the highest, whereas the level of P-gp in SPCA-lwas the lowest. The chemosensitivity to cisplatin, doxorubicine and VP-16 in four cell lines were different. Both doxorubicin and Cisplatin were the most sensitive compounds to SPCA-1; VP-16 was the most sensitive compound to SK-MES-1. There was a positive correlation, between GST-πexpression and resistance to cisplatin, between TOPOⅡαexpression and resistance to VP-16; and a negative correlation between TOPOⅡαexpression and resistance to doxorubicin. Conclusively, different endogenous levels of P-gp, MRP, LRP, GST-πand TOPOⅡαin human lung cancer cell lines may suggest various chemosensitivity to cisplatin, doxorubicine and VP-16.2.Compared to parental cell line A549, the expression of P-gp and MRP enhanced and TOPO II a level decreased in cisplatin resistance cell line A549/DDP.3. The expression of P-gp at both mRNA and protein level can be obviously inhibited by verapamil in all the four cell lines. With the pretreat-ment of verapamil, NCI-H-446 was more sensitive to cisplatin; SPCA-1, NCI-H-460 and NCI-H-446 were more sensitive to VP-16 as well compared to the control group.4. We found that the endogenous levels of GRP78 in four cell lines vary, with NCI-H-460 was the highest. Compared to the control group, the expression of GRP78 and GRP94 at protein and GRP78 mRNA level with the A23187 pretreated was significantly increased in the four cell lines. The IC50 of VP-16 also showed significant elevation for the cells induced by A23187. There was a positive correlation, between endogenous levels of GRP78 and resistance to VP-16. With the pretreatment of A23187, the protein level of GRP78 and GRP94 was related to the resistance to VP-16 in four cell lines. And mRNA level of GRP78 was associated with the resistance to VP-16 in SK-MES-1, SPCA-1 and NCI-H-446 cell lines.5. Both drug resistance-related proteins and GRPs showed obviously basal expression in all four types of lung cancer cell lines. Except for P-gp，the other four drug resisitance-related protein exhibited similar expression tendency to GRP78 and GRP94 in all the fours cell lines.. There was a positive correlation between GRP78 and LRP(r=0.773, P<0.05).Conclusions:1. Co-expression of P-gp, MRP, LRP, GST-πand TOPOⅡa may play an important role on the intrinsic resistance in lung cancer, meanwhile the different expression in four histopathological subtypes of lung cancer cell lines may cause the various resistance to anticancer drugs. GST-πmay be significant for prediction of the intrinsic resistance to cisplatin, whereas TOPO II a may be a valuable protein for estimating the intrinsic resistance to doxorubicine and VP-16.2. P-gp, MRP and TOPOⅡαmay be important factors for the acquired resistance to cisplatin in lung cancer3. Down-regulation of P-gp is associated with the intrinsic resistance to cisplatin in NCI-H-446 cell line and to VP-16 in SPCA-1, NCI-H-460 and NCI-H-446 cell lines.4. Various chemosensitivity to VP-16 may be resulted from their different endogenous levels of GRP78 in human lung cancer cell lines. Expression of GRP78 and GRP94 at protein level is associated with the intrinsic resistance to VP-16 in all four cell lines, while mRNA expression of GRP78 is related to the resistance to VP16 in SK-MES-1, SPCA-1, NCI-H-446 cell lines.5. Both drug resistance-related proteins and GRPs are interacting important factors involving in lung cancer chemoresistance. Therefore, combined detection of two groups proteins can not only contibute to confir-ming chemoresistance mechanism and evaluating therapeutic effect, but also offer effective and rational guidelines in lung cancer chemo-treatment.更多还原