【作者】 杨啊晶；

【导师】 李电东； 何琪杨；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2008， 博士

【摘要】 力达霉素（Lidamycin,LDM）是烯二炔类抗肿瘤抗生素,由本所独立开发,目前正在进行二期临床试验。以前的研究表明:LDM的作用方式独特,高浓度引起肿瘤细胞非caspase依赖的凋亡,而在较低浓度下,引起细胞具有衰老样表型的有丝分裂死亡。然而,肿瘤细胞对力达霉素不同反应的机制至今仍未阐明。本研究从细胞存活分子方面入手,利用不同的敏感和耐药细胞来探讨其作用机制。首先,Western blot、流式、SA-α-gal染色以及染色质凝集实验的结果表明:不同浓度的LDM引起人肝癌BEL-7402细胞出现2种特征的死亡方式。10nM LDM作用细胞24h后,出现典型的凋亡特征,p53蛋白的表达明显增高,出现PARP、SIRT1的切割片断以及亚G₁峰。而0.5nM LDM作用细胞后,出现具有衰老样表型的有丝分裂性细胞死亡。细胞先被阻断在G₂/M期,然后SA-β-gal染色阳性率明显增高（72h）,此时,p53蛋白的表达没有明显的变化,存活分子SIRT1也一直维持表达。此外,使用Western blot检测其他存活分子的表达变化。10nM LDM作用细胞后,不同时间收集细胞至24h,P38的磷酸化水平明显升高,FOXO3a和Bim的表达均于给药后下降,MnSOD与Cu/ZnSOD的表达未见增高。而在0.5nM LDM作用组中,FOXO3a和Bim的表达均于给药4h开始到72h逐渐增加,MnSOD与Cu/ZnSOD则于给药4～8h内一过性增高,Akt被明显激活的时间长至48h。这些研究表明:存活分子影响LDM的活性,导致出现不同的细胞死亡方式。使用染色质凝集和DNA凝胶电泳的方法,观察到1μM LDM作用1h可切割MCF-7细胞的DNA,但是却不能切割MCF-7耐多柔比星细胞的DNA。Western blot的结果表明这种现象与LDM迅速降低MCF-7细胞（而非MCF-7/DOX细胞）PARP和P53的含量有关,而与P糖蛋白、谷胱甘肽以及存活分子如SIRT1和Akt无关。同时,caspase抑制剂不能抑制LDM降低MCF-7细胞PARP和P53的作用。10nM LDM作用p53野生型（p53 wt）和p53敲除型（p53 ko）人结肠癌HCT116细胞24h,MTT结果表明p53 wt比p53 ko HCT116细胞对LDM更敏感。其机制与SIRT1和Akt有关。同时LDM诱导HCT116细胞出现衰老样表型、G₂/M期阻滞、染色质快速凝集现象。LDM对人正常肝L-02细胞和肝癌BEL-7402细胞的MTT结果表明:LDM对L-02细胞的生长抑制作用要低于BEL-7402细胞。Western blot的结果表明在未经药物处理的BEL-7402细胞和L-02细胞中,PARP的表达未见明显差异;与L-02细胞相比,BEL-7402细胞的SIRT1和P53表达水平较高,而Akt的表达水平较低。本研究使用不同类型的细胞,初步证明存活分子影响LDM在肿瘤细胞内的活性而出现不同类型的死亡方式,有助于解释LDM的作用机制。更多还原

【Abstract】 Lidamycin（LDM） as a member of the enediyne antibiotics family is developed independently by our institute,and it has been in phaseⅡclinical trial.Previous studies showed that high concentrations of LDM induced tumor cells to non-caspase dependent apoptosis,however,LDM at lower concentrations induced mitotic death with senescence-like phenotype（SLP）.The mechanism of different responses to tumor cells for LDM is still unknown.Using different sensitive and drug-resistant tumor cells,we examine the effect mechanisms from cell survival molecules profile.Firstly,the results of Western blot,FACS,SA-β-gal staining and chromatin condensation experiment indicated that different concentrations of LDM could induce two modes of death characteristic in human hepatoma BEL-7402 cells.The cells treated with 10 nM LDM for 24h appeared classic apoptotic characteristics,such as obvious increase of p53 protein,cleavaged fragments of PARP and SIRT1 as well as sub-G₁ peak.However,the cells treated with 0.5 nM LDM could induce mitotic death with SLP. The cells were arrested in G₂/M phase firstly,then the percentage of the cells with senescence-associatedβ-galactosidase activities（72h） was increased obviously.At the same time,the expression of p53 protein was no obvious change,and the survival molecule SIRT1 also sustained its expression.In addition,we also detected the expression changes of other survival molecules by Western blot.The cells were treated with 10 nM LDM for 24h and collected at indicated time points.The results showed that the phosphorylation level of p38 protein was obviously increased,and the expression of FOXO3a,Bim was declined after LDM treatment.The results also indicated that the expression levels of MnSOD and Cu/ZnSOD was not increased.At 0.5 nM LDM treatment group,the expressions of FOXO3a and Bim were gradually increased from 4h to 72h after treatment.MnSOD and Cu/ZnSOD were transient increased within 4～8h and Akt was activated obviously until 48h.These studies indicated that survival molecules influenced the action of LDM and resulted in different cell death modes ultimately.The genome DNA cleavage after 1μM LDM treatment for 1h was observed in MCF-7 cells by chromatin condensation and DNA electrophoresis,but did not observed in doxorubicin-resistant MCF-7 cells.This phenomenon was related with rapidly decrease of PARP and P53 in MCF-7 cells,but not in MCF-7/DOX cells detected by Western blot.Moreover,it was not related with P-glucoprotein,glutathione and prosurvival molecules such as SIRT1,Akt.Furthermore,the caspase inhibitor did not inhibit this effect of LDM.The inhibition of proliferation in human p53 wild-type and p53 knocked out colorectal HCT116 cells treated with 10 nM LDM for 24h assayed by MTT showed that p53 wt HCT116 cells was more sensitive to LDM induced toxicity than p53 ko HCT116 cells.The mechanism within it might be related with SIRT1 and Akt.Furthermore, LDM could induce the cells appearing SLP,G₂/M arrest and chromatin condensation.The result of MTT between human liver normal L-02 cells and human hepatoma BEL-7402 cells treated with LDM showed that the toxicity to L-02 cells was lower than to BEL-7402 cells.The result of Western blot indicated that the expression level of PARP was no obvious difference between untreated L-02 cells and BEL-7402 cells.But, compared with L-02 cells,the expression levels of SiRT1 and P53 were higher and the level of Akt was lower in BEL-7402 cells.This study demonstrated initially that prosurvival molecules influenced the action of LDM in tumor cells and resulted in different modes of cell death by using various types of cells.And it is helpful to explain the mechanisms of LDM.更多还原