【作者】 王旭；

【导师】 王冠军；

【作者基本信息】 吉林大学 ， 内科学， 2007， 博士

【摘要】 肿瘤本身是一种多因素、多步骤、多基因改变的疾病,每种药物均只能针对一种或几种靶分子起作用,如果将多种抑制剂联合应用可能会使药物发挥更好的效果,联合用药将成为医学肿瘤学的发展趋势。在美国举行的ASCO会议提出了靶向治疗,并提出了联合用药和应用多靶向药物的概念,这会比前几代药物更有效,产生更好的效益。有关肿瘤的发生发展机制到目前为止已经有很多说法,最近对于基因表型修饰以及信号传导等的研究成为热点。苯丁酸钠SPB(Sodium phenylbutyrate)是去乙酰化酶抑制剂(HDACI Histone deacetylase inhibitor),吉非替尼(ZD1839,Gefitinib)是一种选择性EGFR酪氨酸激酶抑制剂。通过对两种药物作用机理分析后,推断联合应用这两种药物可能通过以下途径起协同作用:①加强肿瘤细胞的周期抑制,使细胞阻滞在G1期;②抑制肿瘤周围血管生成和抑制肿瘤细胞的转移;③调节与肿瘤增殖、周期相关蛋白如P21WAF1、P53和CDK4/CyclinD1蛋白的表达,④协同抑制细胞内的信号传导。再加上我们对肿瘤细胞的筛选的前期工作,我们认为两种药物有其合用的必要性和理论依据。本研究首次发现了高浓度Gefitinib对血液系统的肿瘤也有一定的抑制作用,血液系统肿瘤细胞属于EGFR不表达或者微表达细胞,说明了Gefitinib不仅仅通过抑制EGFR这一条途径起作用的,除了抑制EGFR还可能抑制其他的信号传导途径。本研究首次发现了SPB和Gefitinib对SMMC-7721和肺癌A549细胞是通过抑制P38MAPK信号传导通路来抑制细胞增殖的,两药各自是通过阻滞细胞周期在G1期而抑制增殖的,同时依赖于P53的调节,并呈剂量和时间的依赖性。本研究首次应用体内体外实验两个角度观察合用SPB和Gefitinib的抑制肿瘤细胞的增殖效应,结果观察体外实验联合用药对A549细胞和SMMC-7721抑制作用明显,其机制是通过依赖P53的P38MAPK信号传导途径来诱导P21表达增强,CyclinD1/CDK4复合物表达下降及G1周期阻滞来实现的。体内实验也可以看到联合用药的明显作用,肿瘤重量体积增殖明显抑制,小鼠存活率和生存时间明显提高,与单独用药组相比,差异具有显著性。我们的体外体内实验从用药浓度,用药时间,以及从蛋白表达的水平和信号传导途径等角度系统的论证了联合应用SPB和Gefitinib两种药物对几种细胞株的抑制作用,并阐明其合用的信号传导方面的机制,为下一步临床上的应用奠定了理论基础。更多还原

【Abstract】 Tumor is a kind of disease that multiple factors、multiple steps and mulitiple genes changed,each medicine can play a part in only one or several target, if we can combined multiple inhibitors of tumor will get better effect, drug combination will become a tendency that tumor curement object.The ASCO meeting holded in US draw a new viewpoint that target treatment、drug combination and multiple target medicine combination et al.Such method will bring better effect and profit.The mechanism promote the tumor growth and carcinogenesis will uncertain to date, the gene phenotype modify and signal transduction have been study hot about the mechanisms of tumor growth and carcinogenesis.The gene phenotype modify can control the activation and inactivation of function gene so that activate selectively the specific hereditary information .Many studies have certified that tumor cells have the abnormality of gene phenotype states, the modify of acetylation and deacetylation is most major way that modify gene phenotype.Such sytle paritipate in the development and occurrence of many tumors. The mechanism that HDACIs cure tumor is a Hotpoint that interven the gene phenotype modify, summarize previous studies, we sum up several mechanism of HDACIs 1: induce the differentiation of tumor; 2: chang the cycle or tumor through impacting the P21WAF1、P53、Rb、myc et al that related to the cell cycle;3:induce the apoptosis of tumor;4:up-regulate the MHCⅠ、Ⅱ、CD40 molecules to regulate the immune function;5:suppress the vascularization of tumor ;6:revers the inverted phenotype;7:activat the silent gene;8:recover the sensitivity of drug-resistant tumor cells to drugs.The moleculer mechanism about the carcinogenesis ,the abnormality of signal transduction mediated by epidermal growth factor receptor is related to the development and occurrence of tumor.Many evidences have unclose that the overexpression of EGFR in many tumor coming from epithelium tissue,the activation of EGFR can activate the Ras/Raf/MAPK、PI-3K/Akt et al signal transduction pathway,in the end,such signal pathway influence the gene expression to induce the proliferation and differentiation biological effect.Gefitinib is a kind of micromolecule compound that selectively suppress the EGFR kinase,it can bind competivly the EGFR kinase to suppress the activation of EGFR kinase,it can block the signal transduction mediated by EGFR to suppress the proliferation and metastasis and induce the apoptosis of tumor,Gefitinib can specificly suppress the HER1 when low concentration ,it can suppress other PTK activation when high concentration ,so it has repressed activity to a series of tumor lines. Frist,we screen the optimal concentration of SPB and Gefitinib in vitro,1.0mM and higher concentration SPB have obviously suppression effect to three kinds of hametology lines (U937、HL-60、K562),such result consists with the effect on the tumor origined from epithelium tissue treated by SPB, compared with tumor lines origined from epithelium tissue ,the effect of hametology cell lines treated by SPB is more sensitive.The survival rate and the cell counts in U937、HL-60、K562 cell lines treated by Gefitinib only over 30g/L concentration show significant difference compared with control group,but the survival rate is over 75%,the cells number loss isn’t obvious when the concentration is increased to 50g/L,the effect isn’t consistant with the result in the cells origined from epithelium tissue treated by Gefitinib. The repressed effect is obviously when the concentration of Gefitinib was increased to 40g/L in the cell lines origined from epithelium tissue.We get the optimal drug concentration which suppression rate is obvious but survival rate is over 75%,as well as the conclusion that SPB is sensitive to hametology cells,but the Gefitinib is sensitive to cells line origined from epithelium tissue,we even found the synergistic inhibition between Gefitinib and SPB when A549 and SMMC-7721 treated.Interestingly,the Gefitinib is the inhibitor of EGFR,however,the EGFR is specific expressed in the cell origined from epithelium tissue,but it can suppress the proliferation of U937、HL-60、K562 cells though not so strong.Such result consist with conclusion that high concentration Gefitinib can suppress the other PTK activity in published literature ,morever other unusual mechanisms,It discover the that the gefitinib suppress the cell proliferation through not only RGFR pathway ,it can explain why low concentration Gefitinib cannot suppress the proliferation of hamtology cell lines.We conclude the possible mechanism that combining such two drugs through analyzing such two drugs mechanism of action:①enhance the arrest the cycle ,arrest the tumor cell in G1 stage;②suppress the angiogenesis around the tumor tissue and metastasis;③regulate the expression of P21WAF1、P53、CDK4 gene and protein that related to the apoptosis and cycle;④synergistic inhibit the signal transduction .We consider the essentiality and rationality to combining such two drugs according to the analysis and previous expriment experience. P38MAPK is a family member in the mitogen-activated protein kinase, MAPK cascade is a predominant signal transduction system in cells, it adjust the growth、differentiation、cleavage、death and function synchronization among cells process.We detected the expression of phospho-P38MAPK and gene、protein and apoptosis when suppress the P38MAPK activity with Westernblot assay to determine if the drug combination of SPB and Gefitinib can adjust the survival state through the P38MAPK signal transduction pathway.We found the drug combination of SPB and Gefitinib can get better effect in tumor cell origined from epithelium tissue than hematological cells,the effect of drug combination isn’t so obvious in hematological cells compared with SPB or Gefitinib only(P>0.05).So we chose two kinds of cells including the A549 and SMMC-7721 cell lines to determine the action and mechanism of drug combination in vitro.We found the drug combination of SPB and Gefitinib can increase the apoptosis rate of SMMC-7721 and A549 cell, compared with SPB or Gefitinib only,the difference shows significant(P<0.05), but the extent of SPB and gefitinib under optimal concentration induced apoptosisisn’t so obvious,drug combination increase only the apoptosis rate from 7-8% to 11-12% compared with SPB and Gefitinib only,it elucidate that the mechanims of the drug under optimal concentration is suppression of proliferation but not inducing apoptosis of tumor cells. SPB and Gefitinib can both arrest such two kinds of tumor cells in G1 stage to suppress the proliferation of cell.The drug combination of SPB and Gefitinib can arrest cycle in G1 stage obviously compared with SPB and Gefitinib only(P<0.05). SPB and Gefitinib only can also enhance the expression of P53 and P21 protein, suppress the expression of CDK4/CyclinD1. The expression tendency of these protein is more obvious in drug combination group compared with SPB and Gefitinib group(P<0.05),the expression of P53﹑P21 gene and protein decreased, CDK4/CyclinD1 increased when the P38MAPK kinase activated by P79350, such expriment delinate the drug combination of SPB and Gefitinib increase the expression of P53﹑P21,decrease the CDK4/CyclinD1 compound though P38MAPK signal transduction pathway.It certifies that P38MAPK signal transduction pathway affect te proliferation mediated by cell cycle,we can adjust the change of the cycle an apoptosis through changing the related gene and protein.Different literatures have different viewpoints about the influnce on the gene and protein by SPB and Gefitinib,some thought the SPB adjust cell cycle independ on P53,some thought Gefitinib can arrest G2/M stage.Different studies draw different conclusions because of different drug concentrations and cell lines.Such two drugs are both concentration and time dependent style according to previous literatures.Our results consist with above conclusions,some studies thought drug combination of HDACI and Gefitinib is sequence-dependent style,we don’t testify such conclusion,we detected the suppressed effect that SPB 12h prior to Gefitinib, such partly consisted with published literatures.To authenticate the suppressed effect after metabolism and the influnce by immunization and endosecretory ,we chose the nake mices injected from right armpit as object in vivo.We chose concentration that screened in preliminary experiment,the SPB and Gefitinib only can suppress the preliferation of human A549 cell line in vivo,they can suppress tumor weight and volume in that nick mice that tumor tissue has formed, moreever lengthen the survival time and increase the survival rate .We detected the expression of P53 and CDK4 protein with immunohischemistry methods,drug combination can enhance the expression of such protein ,such consist with the results in vitro,we also detected the suppressed effect with the drug concentration according to related published literatures.We didn’t screen the concentration grade in vivo and compare the effect under the same concentration between in vitro & vivo, we need to investigate the experiment further.更多还原