【作者】 韩菲菲；

【导师】 甄永苏； 邵荣光； 李电东；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2010， 博士

【摘要】 细胞中受到外界DNA损伤物质的侵害后会激活自身修复DNA的途径来进行DNA的损伤后修复,这是许多诱导DNA损伤药物发生耐药或剂量升高的重要原因之一。通过降低细胞内部的DNA损伤后修复水平来增敏DNA损伤药物是寻找放疗、化疗增敏剂可行的方法。细胞在接受DNA损伤因素的作用后是否能够存活主要取决于DNA双链断裂程度。ATM/ATR等PIKKs成员在应答DNA损伤、维持基因组稳定性过程中扮演重要的角色,它们传递基因组损伤的信号、阻滞细胞周期进程并且促进DNA修复。ATM主要对DNA双链断裂进行应答,下游最重要的底物是Chk2；ATR对DNA单链断裂、交联或者复制叉进行应答,下游的重要底物有Chk1和BRCA1等。Chk1和Chk2都将细胞阻滞在G2／M期,抑制DNA复制,促进受损的DNA进行修复。力达霉素(lidamycin, LDM;又名C-1027)是烯二炔类(enediyne)抗肿瘤抗生素家族的成员之一,通过诱导DNA断裂可以达到强烈的肿瘤细胞杀伤作用,在中国正在进行Ⅱ期临床试验。作为一种DNA损伤药物,力达霉素同样能够激活细胞内的DNA损伤修复通路,这可能是影响其疗效的主要原因之一。Hsp90是人们非常关注的治疗肿瘤的一个靶点,目前有多种Hsp90抑制剂正在进行临床试验,单独的Hsp90抑制剂并不能发挥很好的效果,但研究中发现Hsp90抑制剂与很多种化疗药物联合应用效果良好,能够起到增敏的作用。关于Hsp90抑制剂增敏化疗药物的机制众说纷纭,可能根据化疗药物的不同,Hsp90所抑制的通路所起的作用也不尽相同。其中许多研究证实Hsp90抑制剂可以降低基因组的稳定性。本研究主要研究了Hsp90抑制剂格尔德霉素及其衍生物CNDG与力达霉素联合给药后对力达霉素在人卵巢癌SKOV-3细胞中的增敏作用,观察联合给药后细胞的生长、增殖、DNA损伤水平,探讨Hsp90抑制剂对力达霉素增敏的作用机制。Hsp90抑制剂我们选择了典型的格尔德霉素(Geldanamycin, GDM)和由本实验室合成的格尔德霉素衍生物CNDG。一、力达霉素对卵巢癌细胞的影响MTT检测表明,LDM (0.01nM、0.05nM、0.1nM、0.5nM和0.1nM)作用于SKOV-3细胞24小时后,可以呈剂量依赖性的抑制细胞增殖,细胞凋亡比率随浓度增加而升高。Western blot检测显示0.01nM的力达霉素可以增强HER2的磷酸化水平,而0.1和1 nM的力达霉素降低HER2的磷酸化。在体外模拟缺氧条件用CoC12处理细胞体外模拟肿瘤的缺氧状态,结果显示力达霉素可以明显的降低缺氧诱导因子HIF-1α的水平。二、Hsp90抑制剂可以增强力达霉素对卵巢癌SKOV-3、乳腺癌MCF-7、肺癌A-549、肝癌Bel-7402细胞的杀伤作用用MTT法检测力达霉素与格尔德霉素(GDM)联合应用对卵巢癌SKOV-3细胞增殖的影响,计算力达霉素(0.01nM、0.05nM)与格尔德霉素(10nM、50nM,100nM、500nM, 100nM)单药及联合后对卵巢癌SKOV-3细胞增殖的抑制作用,并通过公式计算其联合指数,力达霉素与格尔德霉素100nM、500nM联合作用有明显的联合抑制细胞增殖的作用。CNDG与力达霉素联合可显示与格尔德霉素相似的作用。实验表明力达霉素与Hsp90抑制剂联合应用可以明显增强抑制细胞增殖的水平。同样的实验条件,我们选择了三种其他种类的肿瘤细胞进行相应的实验,Hsp90抑制剂同样能够增强力达霉素对细胞的杀伤能力,伴随着诱导凋亡的增加、DNA损伤后修复的减少,G2／M期阻滞降低等现象,说明Hsp90抑制剂对力达霉素的增效作用是可以广泛适用于多种肿瘤细胞。三、Hsp90抑制剂增强力达霉素对SKOV-3细胞诱导凋亡的作用以不同浓度格尔德霉素预处理SKOV-3细胞16小时,力达霉素(0.01nM, 0.05 nM)作用于24 h后,PARP的切割水平在联合应用时大幅度升高；用Annexin V-PI双染检测凋亡细胞,其中0.05 nmol/L LDM作用于SKOV-3细胞凋亡率为14%,200nM格尔德霉素作用于SKOV-3细胞凋亡率为16.1%,两者联合后细胞的凋亡比例为32.2%,单独用药与联合用药相比P<0.01。联合指数0.89。四、Hsp90抑制剂增强力达霉素对卵巢癌SKOV-3细胞的DNA双链断裂作用力达霉素杀伤肿瘤最主要是通过切割DNA,导致DNA双链断裂。我们借助γH2AX水平来了解细胞内DNA双链断裂的程度,通过Western blot和免疫荧光实验可以看到,γH2AX在力达霉素和格尔德霉素联合应用的情况下细胞内的γH2AX明显增加,尤其是细胞核内的γH2AX水平,说明格尔德霉素的联合应用能够增强力达霉素的DNA断裂能力。五、Hsp90抑制剂增强力达霉素对卵巢癌SKOV-3细胞的杀伤作用是通过抑制力达霉素诱导的DNA损伤后修复实现的在用0.05 nM力达霉素处理细胞1小时后,细胞内的γH2AX大量增加,说明在此期间细胞内产生大量的DNA双链断裂,但6小时后yH2AX阳性细胞减少或细胞内γH2AX染色程度减弱,细胞内本来存在的双链断裂减少了；而用格尔德霉素预处理的细胞内的γH2AX没有发生大的变化,所以格尔德霉素的预处理可以抑制由力达霉素诱导的DNA损伤后修复。六、Hsp90抑制剂可以抑制力达霉素诱导的G2／M期阻滞不同剂量力达霉素处理SKOV-3细胞24 h,流式细胞仪检测细胞周期分布变化。结果显示,力达霉素诱导SKOV-3细胞发生G2／M期阻滞,该作用呈剂量相关。随着力达霉素浓度增加,G2／M期细胞比例逐渐增加。用不同剂量的格尔德霉素预处理细胞16小时,再用不同剂量的力达霉素处理细胞24小时,用流式细胞仪检测细胞周期分布。在SKOV-3细胞中,0.05 nM力达霉素处理细胞可以使G2／M期细胞从15.28%增加到76.79%,而经过200nM格尔德霉素预处理细胞G2／M期百分比大大下降,从76.79%下降到29.33%。说明格尔德霉素可以抑制力达霉素在SKOV-3细胞中诱导的G2／M期细胞周期阻滞。七、Hsp90抑制剂可以降低某些与基因组稳定性相关的蛋白或抑制其活性Western blot分析Hsp90抑制剂可以抑制多种与基因组稳定性相关的蛋白水平或抑制其活性,其中包括ATM、ATRIP、BRCA1、RPA70。其中,ATRIP的总蛋白水平的下降可以被蛋白酶体抑制剂MG132的作用所阻断。结论：本研究首次研究了Hsp90抑制剂对烯二炔类抗生素力达霉素的增敏效果。在卵巢癌SKOV-3细胞中Hsp90抑制剂能够通过抑制DNA损伤后修复增加力达霉素对DNA的断裂作用,从而增强力达霉素的细胞毒作用。同时在其他种类的细胞：人乳腺癌MCF-7、人肺癌A549和人肝癌Bel-7402中也可以看到相类似的作用。总之,Hsp90抑制剂可以降低细胞基因组稳定性,抑制力达霉素所诱导的DNA损伤后修复作用,增加力达霉素对细胞造成的DNA损伤,从而增强其抗肿瘤活性。更多还原

【Abstract】 Potency of Lidamycin on ovaraian carcinoma suppression and Hsp90 inhibitors Geldanamycin and its analogue CNDG enhance the cytotoxicity of lidamycinTumor resistance to DNA damaging agents is often associated with enhanced DNA repair activity. Reducing DNA damage repair is proved to be an effective way to sensitize cancer cells to DNA damaging agents. Enediyne lidamycin (LDM, also called C-1027) showed extremely potent cytotoxicity by inducing DNA damage and Hsp90 is an interesting target for anticancer treatment recently. In this research we investigated whether Hsp90 inhibitors, geldanamycin (GDM) and its analogue CNDG, could further enhance the potent cytotoxicity of LDM. The survival of cells after DNA double-strand breaks (DSBs) is heavily dependent on the DNA damage repair. Phosphatidylinositol-3 kinase-related kinases (PIKKs) members such as ATM and ATR play pivotal roles in response to DNA damage and maintaining genome integrity. They transduce genomic stress signals to halt cell cycle progression and promote DNA repair. After DNA damaging agent treatment PIKKs is activated to attempt the cell cycle arrest and DNA damage repair. ATM responds mainly to DSBs, whereas ATR is activated by single stranded DNA and stalled DNA replication forks. Further, the activation of ATM and ATR triggers G2/M phase arrest and some DNA repair enzymes. Chk2 can be directly activated by ATM and Chkl by ATR. Both Chkl and Chk2 can arrest cell cycle at G2/M phase, retarding DNA replication and stimulating the repair of damaged DNA.Lidamycin (LDM) is an enediyne antitumor antibiotic produced by Streptomyces globisporus C-1027. It is currently being evaluated in phaseⅡclinical trials as a potential chemotherapeutic agent in China. LDM induces DSBs leading potent cytotoxicity and marked inhibition of tumor growth in vivo. As a DNA damaging agent LDM induced DNA damage repair and this might decrease the effect of LDM.Hsp90 is important for stabilization and trafficking of tyrosine and serine/threonine kinases that are activated in response to genotoxic stress, including those that are essential for survival of cancer cells. GDM is an ansamycin antibiotic that inhibits HSP90 by binding to the NH2-terminal ATP binding domain, leading to degradation of HSP90 clients. Some Hsp90 inhibitors are in phaseⅠ/Ⅱclinical trials in combination with radiation or other chemotherapeutic agents. Many reports show that Hsp90 inhibitors enhance the effect of DNA damaging agents, and some of them indicate that Hsp90 inhibitor decreases DNA damage repair through inhibiting ATM dependent DNA damage repair.In this report Hsp90 inhibitors GDM and its analogue CNDG were found to enhance the anticancer effect of LDM. Our results suggested that GDM pretreatment potentiated the cytotoxity of LDM by decreasing the LDM-induced DNA damage repair. To our knowledge, it is the first report about Hsp90 inhibitor enhancing potent cytotoxicity of LDM or enediyne antitumor antibiotic.1. LDM effects on SKOV-3 cellsBy MTT assay, LDM inhibited the proliferation of SKOV-3 cells. Examined with Annexin V-FITC/PI double staining flow cytometry, the apoptotic rates in 0.01 nM and 0.05 nM LDM treated cells were 10.92% and 44.78% respectively; at 0.05 nM, LDM dramatically increased the comet tail; and 0.1 nM of LDM inhibited the phosphorylation of HER2. In the hypoxia condition, LDM reduced the level of HIF-1α.2. GDM and its analogue sensitize SKOV-3, MCF-7 and Bel-7402 cells to LDMIt has been demonstrated that inhibition of Hsp90 could potentiate the efficacy of ionization radiation, antimetabolites and alkylating agents. So we investigated whether inhibition of Hsp90 could potentiate the cytotoxicity of LDM. MTT assays were used on different cancer cells. Cells were exposed to LDM for 24 h after 16 h preincubated with GDM. Under our experimental conditions, low concentrations of LDM (0.01 and 0.05 nM) did not show extensive growth inhibition of SKOV-3 cells. In the following experiments, we chose 0.01 and 0.05 nM LDM for SKOV-3 as appropriate doses to detect the potentiation effects of Hsp90 inhibitor.By MTT assay, LDM markedly inhibited cell growth by inhibiting Hsp90. Combined with 100 and 500 nM GDM,0.01 and 0.05 nM LDM induced more growth inhibition than LDM alone. We compared the cytotoxicity of the GDM-LDM combination to the effect of the two agents alone using the median effect method, which determines whether the cytotoxicity for the combination is greater than (CI＜1), equal to (CI=1), or less than (CI>1) the additive effect of the individual agents. Under the indicated doses, most of the CI values were less than 1 and some of them were less than 0.7. These results suggested that the combination of LDM and GDM showed a synergistic effect in SKOV-3 cells. Notably,17-(6-cinnamamido-hexylamino)-17-demethoxygel- danamycin (CNDG), a derivate of GDM synthesized in our laboratory had similar protentiation effect on LDM cytotoxicity to cancer cells. Under the same condition, we also chose Bel-7402, A549 and MCF-7 cells to check this combination method, and the results were quite similar.3. GDMpotentiats the apoptosis-inducing effect of LDMWestern blot analysis was used to detect the cleavage of PARP, an indicator of caspase-mediated apoptosis. The 89-kDa cleaved fragment of PARP increased dose-dependently in SKOV-3 cells exposed to LDM. Enhanced levels of cleaved PARP appeared after combination treatment.To confirm the augmentation of LDM-induced apoptosis by GDM, we performed annexin V/PI staining assay. Consistent with above results, LDM in combination with GDM pretreatment induced more apoptosis than LDM alone. The apoptosis cell ratios were increasing from 14% to 32.2%.4. GDM enhances LDM-induced DNA damageTo examine the effects on DNA damage induced by LDM in the absence and presence of GDM, the phosphorylation of Ser-139 of histone H2AX was compared by immunoflurorescence and Western blot. The treatment of LDM or GDM alone did not increase yH2AX protein, however, the combination of GDM and LDM markedly enhanced yH2AX levels. The figure of yH2AX foci by immunoflurorescence also confirmed the result.The alkaline comet assay could provide a direct measure of DSBs. There was no significant DNA damage in SKOV-3 cells exposed to either LDM or GDM alone. Significantly more DNA damage in combination-treated cells versus either LDM or GDM alone treated cells.5. GDM inhibits LDM-induced DNA damage repairyH2AX levels after were determined 1 h and 6 h LDM treatment. LDM increased the number of yH2AX positive cells. However, the number of yH2AX positive cells was reduced dramatically after 6 h treatment as compared with 1 h treatment, indicating that damaged DNA was partially repaired. The combination of GDM and LDM did not seriously affect the yH2AX positive cell number compared with LDM alone at 1 h treatment. Notably, after 6 h treatment, the percentage of yH2AX positive cells was significantly increased in the combination-treated cells as compared with LDM alone. Time course analysis confirmed that GDM reduced DNA damage repair in LDM-treated cells.6. GDM abrogates LDM-induced G2/M arrest and phosphorylations of Chkl and Chk2Most of DNA damage agents including LDM induce G2/M phase arrest. Treated by 0.05 nM LDM cells at G2/M phase were increased from 15.28%(control) to 76.79%. At the same condition,200 nM GDM did not induce G2/M arrest significantly. However, the pretreatment of GDM down-regulated the number of G2/M phase cells, and the percent of G2/M phase in LDM-treated cells was decreased from 76.79% to 29.33%. By Western blot analysis the phosphorylations of Chkl and Chk2 induced by LDM were decreased by pretreatment of GDM.7. GDM compromised LDM-induced ATM activity, the protein level of ATRIP and DNA damage related proteinsTo address the molecular mechanism involved in GDM mediated inhibition of DSB repair, we focused on LDM-induced ATM and ATR activity. As shown, ATM phosphorylation was increased within 1 h of LDM treatment. By contrast, cells pretreated with GDM had a decrease of phospho-ATM in LDM treated cells. These suggest that GDM interferes with the activation of ATM-mediated DSB repair in response to LDM.ATR is also an important kinase for LDM induced DNA damamge response. Previous studies showed that both ATR and ATRIP played an important role in this pathway, and ATRIP could regulate ATR levels. Thus, we checked ATR and ATRIP level of cells treated with GDM. The results showed that ATRIP dramatically decreased after 16 h GDM-treatment, while ATR had no change. We then examined the states of ATM, ATR and ATRIP in SKOV-3 cells treated with 200 nM GDM for 40 h,0.05 nM LDM for 24 h or with combination, that is, LDM was added after 16 h of GDM pretreatment. The total proteins of ATM and ATR remained unchanged after 16 h GDM. After 40 h GDM pretreatment, the total levels of ATM and ATR were decreased observablely.Then the influence of GDM on ATRIP expression was examined for cells in the presence or absence of proteasome inhibitors. For this study, SKOV-3 cells were incubated for 16 h with 200 nM GDM along with 10μM proteasome inhibitor MG132. The cells were lysed and analyzed for ATRIP protein levels. Evidently, incubation of cells with MG132 abolished the decrease of ATRIP protein in SKOV-3 cells induced by GDM treatment. Also, GDM could decrease other proteins that are related with DNA damage response.In summary, we found that Hsp90 inhibitors could enhance the potent cytotoxicity of LDM. The mechanism was dependent on, at least in part, increasing DNA damage and depressing cell response to DNA damage. Finally, our data suggests that the combination strategy of Hsp90 inhibitors and LDM may represent a novel and promising approach for ovarian cancer treatment.更多还原