【作者】 乔爱敏；

【导师】 吴英良；

【作者基本信息】 沈阳药科大学 ， 药理学， 2006， 博士

【摘要】 EMD-621为人工合成的结构新化合物，本文首次对其体内外抗肿瘤活性进行了较为系统的研究，主要探讨了EMD-621在两种肿瘤细胞：人胃腺癌SGC-7901细胞、人宫颈癌HeLa细胞诱导细胞凋亡的机制；另外还研究了N-去甲基克拉霉素诱导HeLa细胞凋亡的机制。体内实验结果表明，EMD-621对荷S₁₈₀的昆明种小鼠移植肿瘤有一定的抑制作用；体外实验结果表明，EMD-621对人宫颈癌（HeLa），人乳腺癌（MCF-7），人胃腺癌（SGC-7901），人慢性骨髓性白血病细胞（K562）均有明显的细胞毒作用。EMD-621对小鼠成纤维细胞（3T3）也有一定的细胞毒作用，但在等剂量下其毒性远低于顺铂。用形态学方法、核荧光染色、DNA片段化分析、LDH活力测定、细胞周期分布分析及Annexin V-FITC检测磷脂酰丝氨酸（PS）外翻实验充分证实了EMD-621诱导HeLa和SGC-7901细胞死亡机制为细胞凋亡。3.4μmol·L^-1 EMD-621作用于HeLa细胞后，半胱氨酸天冬氨酸酶-3（cysteinyl aspirate specific proteinase-3，caspase-3）和-8的酶原被降解，caspase家族抑制剂、caspase-1、-3和-8的抑制剂可部分抑制细胞死亡，caspase-10抑制剂几乎没有作用，而caspase-9抑制剂则增加了细胞死亡率。Caspase-3底物caspase-3依赖型的DNA酶抑制物（inhibitor of caspase activated DNase，ICAD）和多聚腺苷二磷酸聚合酶（poly-（ADP-ribose）polymerase，PARP）降解，表明EMD-621诱导HeLa细胞凋亡时激活了caspase级联反应，但是caspase-9没有参与其中；死亡受体的配体FasL表达增加，推测EMD-621可能同时诱导FADD（Fas-associated protein with death domain）的表达，激活了下游的caspase途径；促凋亡蛋白Bax（Bcl-2 associated X protein）表达增加，抑凋亡蛋白Bcl-2（B-cell lymphoma-leukemia-2 gene，Bcl-2）表达降低，Bax／Bcl-2表达的比率在药物作用12h后随时间延长而升高，罗丹明123（rhodamine123）染色可见线粒体膜电位下降，表明EMD-621诱导细胞凋亡时激活以线粒体为中心的信号转导途径。细胞凋亡早期p21水平短暂升高，但此后很快被降解；药物作用12h后即可见p53表达增加，并且其表达强度逐渐增加；抗凋亡蛋白SIRT1的表达随着药物作用时间的延长而降低；p38抑制剂SB203580降低细胞的死亡率，细胞外信号调控的蛋白激酶（extracellularsignal-regulated protein kinase，ERK）抑制剂PD98059增加细胞的死亡率，c-Jun N末端激酶（c-Jun N-terminal kinase，JNK）抑制剂SP600125对细胞没有作用；ERK在12h时被抑制，p38在12h时被激活，表明p38发挥了促进细胞凋亡的作用而ERK发挥了促进细胞增殖的作用。EMD-621作用HeLa细胞24h，下调了Ser／Thr激酶Akt的表达，p-Akt先增加后降低，表明Akt参与了EMD-621诱导的HeLa凋亡。蛋白激酶C（protein kinase C，PKC）家族成员在EMD-621诱导HeLa细胞凋亡过程起到促细胞增殖作用。PI3K（phosphatidylinositol 3-kinase）抑制剂wortmannin和PKC家族抑制剂staurosporine均更为显著地增加了EMD-621对ERK蛋白表达及其磷酸化的抑制。以上结果提示我们在HeLa细胞中，ERK对细胞的保护作用可能是依赖上游的PI3K和PKC途径实现的。wortmannin和staurosporine增加了EMD-621对SIRT1表达的抑制，表明SIRT1的失活可能受PI3K家族不同成员以及PKC不同亚型的调控。核内因子κB（nuclear factor-κB，NF-κB）抑制剂PDTC和蛋白消化体（proteasome）抑制剂MG132，增加了细胞的死亡率，同时IκBα也随药物作用时间的延长而被降解，说明NF-κB可能在EMD-621诱导HeLa细胞凋亡过程中发挥作用。酪氨酸激酶在EMD-621诱导HeLa细胞凋亡过程起到促细胞增殖作用。1.6μmol·L^-1 EMD-621作用于SGC-7901细胞12h后，caspase-3酶原被降解，同时ICAD和PARP降解，caspase家族抑制剂、caspase-1、-3和-10的抑制剂可部分增加细胞死亡，而caspase-8抑制剂却显著降低了SGC-7901细胞对药物的敏感性，caspase-9抑制剂几乎没有作用，表明EMD-621诱导SGC-7901细胞凋亡时caspase家族成员起到保护细胞作用。采用ORAC方法考察了EMD-621对活性氧清除的影响，结果提示EMD-621几乎无清除活性氧的能力。加入caspase家族抑制剂后，免疫印迹结果显示PARP仍被降解，进一步证明存在caspase非依赖性蛋白降解了PARP；FasL的表达先短暂增加而后减少，而FADD表达呈时间依赖性减少，推测很可能存在新的作用靶点；药物作用12h后Bax表达显著升高，Bcl-2表达显著下降，细胞色素c的释放也随着药物作用时间的延长而增加，rhodamine123染色可观察到线粒体膜电位下降，表明EMD-621通过改变Bax／Bcl-2的表达，激活以线粒体为中心的信号转导途径而诱导细胞凋亡。药物作用12h后p53表达水平升高，SIRT1表达下调，表明EMD-621能够下调SIRT1蛋白，从而促进了p53和Bax上调。p38、ERK均被激活，p38对凋亡有促进作用，ERK发挥了促进细胞增殖的作用，而JNK几乎没有发挥作用。EMD-621作用SGC-7901细胞12h，下调了Akt的表达，p-Akt也呈时间依赖性减少，表明Akt参与了EMD-621诱导的SGC-7901凋亡。NF-κB抑制剂PDTC和proteasome抑制剂MG132增加了细胞的死亡率，并且IκBα随药物作用时间的延长而被降解，说明NF-κB可能在EMD-621诱导SGC-7901细胞凋亡过程中发挥作用。PKC家族成员以及酪氨酸激酶在EMD-621诱导SGC-7901细胞凋亡过程中均起到促细胞增殖作用。本论文首次发现N-去甲基克拉霉素具有抗癌活性。60μmol·L^-1 N-去甲基克拉霉素作用于HeLa细胞，形态学方法、核荧光染色、DNA片段化分析、LDH活力测定表明N-去甲基克拉霉素诱导HeLa细胞死亡机制为凋亡。实验结果表明药物作用后caspase-3酶原被降解，产生有活性的小片段caspase-3，同时ICAD和PARP降解，DNA片段化，caspase家族抑制剂，caspase-3、-9的抑制剂可部分抑制细胞死亡，表明N-去甲基克拉霉诱导HeLa细胞凋亡时激活了caspase级联反应。Bax／Bcl-2在药物作用后随时间延长而升高，抗凋亡成分Bcl-X_L蛋白的表达不受影响，Cyt．c从线粒体释放到胞浆逐渐增多，表明N-去甲基克拉霉素通过增加Bax／Bcl-2的比例，降低了线粒体膜电位，激活以线粒体为中心的信号转导途径而诱导细胞凋亡。N-去甲基克拉霉素能够下调SIRT1蛋白，从而促进了p53和Bax上调。在N-去甲基克拉霉素诱导的HeLa细胞凋亡中，p38、ERK和JNK均被激活，并且三种MAPK的特异性抑制剂均对凋亡有促进作用，表明它们均发挥了促进细胞增殖的作用。N-去甲基克拉霉素作用HeLa细胞24 h，下调了Akt的表达，p-Akt先增加后开始降低，表明Akt参与了N-去甲基克拉霉素诱导的HeLa凋亡。NF-κB抑制剂PDTC和proteasome抑制剂MG132增加了细胞的死亡率，并且IκBα随药物作用时间的延长而被降解，说明NF-κB可能在N-去甲基克拉霉素诱导HeLa细胞凋亡过程中发挥作用。更多还原

【Abstract】 The mechanisms of EMD-621 -induced cell death in human cervical carcinoma HeLa, gastric adenocarcinoma SGC-901, and mechanism of N-demethyl-clarithromycin-induced HeLa cell death were studied in this dissertation.It showed that EMD-621 had potent antitumor activity in vivo, and it could inhibit the proliferation of SGC-7901, HeLa, K562 and MCF-7 in a dose- and time-dependent manner in vitro. Results of MTT assay, photomicroscopical observation, DNA agarose gel electrophoresis, LDH release, flowcytometric analysis cell-cycle distribution and Annexin V binding to the membrane phospholipid phosphatidylserine showed that EMD-621 could induce cell apoptosis in HeLa and SGC-7901 cells.After treatment with EMD-621, the caspase-3 of HeLa cells was activated, followed by the degradation of caspase-3 substrates, ICAD (inhibitor of caspase dependent DNase) and PARP (poly-(ADP-ribose) polymerase). Caspase family inhibitor, caspase-1, -3, -8 inhibitors could reduce EMD-621-induced HeLa cell death, and the caspase 10 inhibitor almost had no effect, while the caspase-9 inhibitor could increase the cell death. FasL (Fas ligand) and FADD (Fas-associated with death domain) expressions were up-regulated, which suggested that Fas/FasL pathway was activated in EMD-621-induced cell apoptosis. The drug decreased the expression of anti-apoptotic mitochondrial protein Bcl-2, not BcI-X_l, and increased the expression of pro-apoptotic protein Bax. It also could cause the loss of mitochondrial membrane potential. EMD-621 could increase the expression of pro-apoptotic protein p53 and p21 proteins and down-regulated the expression of anti-apoptotic protein SIRT1. The cell death was partially reduced by p38 MAPK inhibitor (SB 203580) and increased by ERK inhibitor (PD98059). At the same time, phosphorylation of ERK was down-regulated and that of p38 was up-regulated indicating different roles of these MAPK at different stages of cell death. Simultaneously, the activation of protein kinase B (PKB/Akt) was also decreased in EMD-621-treated HeLa cell.EMD-621-enhanced engulfment was partially blocked by a nuclear factor (NF-kB) inhibitor PDTC or proteasome inhibitor MG132. Further studies revealed that EMD-621 could induce IκBα(inhibitorκB) degradation. Tyrosine kinase inhibitor genistein also increased the cell death. As a whole, EMD-621 could induce apoptosis in HeLa cells via accumulation of p53, altered Bax/Bcl-2 ratio, activation of caspases and p38 MAPK.In EMD-621-treated SGC-7901 cells, caspase family inhibitor (z-VAD-fmk), caspase-1 inhibitor (Ac-YVAD-cmk), caspase-3 inhibitor (z-DEVD-fmk) and caspase-10 inhibitor (z-AEVD-fmk) partially augmented the cell death, while caspase-8 inhibitor (z-IETD-fmk) reversed the cell death and caspase-9 inhibitor (z-LEHD-fmk) almost had no effect. Caspase-3 was activated followed by the degradation of caspase-3 substrates, ICAD and PARP, and the caspase family inhibitor failed to inhibit PARP cleavage. This result suggested that there might have an unknown signal pathway from the mitochondrial to the downstream protein PARP, which is cleaved in a caspase-independent manner.ORAC (Oxygen Radical Absorbance Capacity) assay was used to detect the change of radical. EMD-621 almost had no effect on scavenging hydroxyl radical. FasL expression was up-regulated in 12 h and began to decrease in 36 h, while FADD expression was down-regulated suggesting that there might exist new target in EMD-621-induced cell apoptosis. EMD-621 up-regulated the expression ratio of Bax/Bcl-2 and significantly increased the expression of p53 protein. It also caused the loss of mitochondrial membrane potential. The cell death was partially reduced by p38 MAPK inhibitor (SB 203580), while ERK inhibitor (PD98059) augmented the cell death; ERK phosphorylation was down-regulated and p38 phosphorylation was up-regulated. Moreover, PI3K inhibitor wortmannin reduced the cell viability, EMD-621 induced IκBαdegradation and tyrosine kinase inhibitor, genistein, also augmented the cell death. Generally, EMD-621 could induce apoptosis in SGC-7901 cells via accumulation of p53, altered Bax/Bcl-2 ratio, and p38/JNK MAPK.N-demethyl-clarithromycin was the main metabolite of clarithromycin in vivo, and it had been reported that N-demethyl-clarithromycin had potent anti-inflammatory activity. There had no report on its anticancer activity. Therefore, for the first time the apoptotic effect of N-demethyl-clarithromycin on human cervical cancer HeLa cells was investigated in this study. This study showed that N-demethyl-clarithromycin could inhibit the proliferation of HeLa cells. Based on morphologic observation, the DNA fragmentation suggested that N-dernethyl-clarithromycin could induce HeLa cell death involved in a mechanism of apoptosis. The caspase inhibitors z-VAD-frnk, z-DEVD-fmk, z-LEHD-fmk effectively enhanced N-demethyl-clarithromycin-induced cell viability, while the z-IETD-fmk almost had no effect. Caspase-3 was activated followed by the degradation of caspase-3 substrates, ICAD and PARE N-demethyl-clarithromycin up-regulated the expression ratio of mitochondrial proteins Bax/Bcl-2 and significantly increased the expression of p53 protein. It also could cause the lost of mitochondrial membrane potential and down-regulated SIRT1 protein expression. ERK, JNK and p38 MAPK were activiated and the cell death were partially increased by ERK inhibitor (PD98059), JNK MAPK inhibitor (SP600125) and p38 MAPK inhibitor (SB203580). Simultaneously, the activation of protein kinase B (PKB/Akt) was also decreased in N-demethyl-clarithromycin-treated HeLa ceils. Moreover, EMD-621 induced IκBαdegradation and tyrosine kinase inhibitor genistein also augmented the cell death. These results indicated that N-demethyl-clarithromycin-induced apoptosis of HeLa cells might involve a mitochondrial-related caspase pathway.更多还原