【作者】 张春玲；

【导师】 吴立军； 池岛乔；

【作者基本信息】 沈阳药科大学 ， 药物化学， 2004， 博士

【摘要】 本文对冬凌草甲素体外抗肿瘤活性进行了较为系统的研究，主要阐述了冬凌草甲素在三种肿瘤细胞：人黑色素瘤A375-82细胞，人宫颈癌HeLa细胞，小鼠纤维瘤L929细胞中诱导细胞死亡的不同机制。 在测定了三种来源血液的肿瘤细胞(人红白血病细胞系，K562；人组织淋巴瘤细胞，U937；人早幼粒白血病细胞，HL-60)和人黑色素瘤细胞，A375-S2；人宫颈癌细胞，HeLa；人乳腺癌细胞，MCF-7；小鼠纤维肉瘤细胞，L929细胞毒时发现三种血液系统的瘤株均对冬凌草甲素敏感，半数抑制浓度分别为：IC50(K562)：16.2±1.2μmol/L，IC50(U937)：27.1±2.3/μmol/L，IC50(HL-60)：20.4±3.6μmol/L，另外三种人源细胞中以A375-S2细胞最敏感，IC50：15.1±1.2μmol/L，对其中鼠源细胞L929较敏感，IC50：35.6±4.2/μmol/L。冬凌草甲素在对敏感细胞株有效浓度下作用于人外周血单核细胞(PBMC)12 h，没有细胞毒作用，但随着浓度的增加，对PBMCs具有较弱的细胞毒作用。 在A375-S2细胞实验中，Hoechst 33258荧光染色后证明34.3 μmol/L冬凌草甲素作用细胞12 h时出现明显的凋亡小体，DNA电泳则表明大剂量的冬凌草甲素(137.4μmol/L)作用细胞12 h时出现坏死特有的弥散泳带。采用流式细胞术及LDH活力测定进一步证明冬凌草甲素诱导A375-S2细胞死亡是平衡于凋亡与坏死之间。合适剂量的冬凌草甲素诱导细胞凋亡。且冬凌草甲素作用细胞12 h时，caspase-3的活力是对照组6.5倍，caspase-9的活力也显著升高。caspase家族抑制剂与caspase-3抑制剂明显抑制冬凌草甲素诱导的细胞死亡率，同时免疫印迹法结果提示caspase-3的底物之一——无活性的PARP蛋白逐渐被消化。以上结果提示caspase-3，-9被激活。在线粒体途径中，用免疫印迹法发现冬凌草甲素改变了促凋亡蛋白Bax与凋亡抑制性蛋白Bcl-xL的表达比率，但对凋亡抑制性蛋白Bcl-2蛋白表达没有影响，同时促进cytochrome c释放到胞浆中。实验结果进一步显示：在冬凌草甲素作用细胞12 h时使肿瘤抑制因子p53蛋白及细胞外信号调节蛋白激酶(ERK)发生磷酸化，却抑制p38激酶的激活，对c-Jun N-末端蛋白激酶——JNK的表达没有影响。继续采用了p53蛋白的上游P13K家族抑制剂——wortmannin与MEK/ERK抑制剂PD98059，考察其对冬凌草甲素诱导A375一52细胞死亡的影响，结果发现:这两种抑制剂均能有效抑制细胞死亡，免疫印迹法发现:PD98059可抑制Bax/Bcl一xL的表达比率的增加及cytochrome。释放，wortnlanin既可抑制p53蛋白磷酸化又可抑制Bax的表达，但对cytochrome。释放没有显著影响。以上结果说明:冬凌草甲素通过改变Bax旧cl一xL的表达比率，进而引起cytochrome。释放，继续启动了下游的casPase一9，一3引起细胞凋亡，这一过程依赖于p53及ERK激活。 在L929细胞实验中，冬凌草甲素没有启动经典的凋亡途径，LDH活力测定表明，诱导细胞坏死的能力强于凋亡，尽管caspaspe家族抑制剂及casPasPe一3抑制剂均增强了L929细胞对冬凌草甲素的敏感性，说明casPases在此信号途径中起保护细胞的作用，通常认为PARp蛋白为casPase一3的底物之一，但在冬凌草甲素的作用12 h pARp一前体被消化激活，且casPaspe家族抑制剂及casPaspe一3抑制剂均不能阻止其降解，反而加强。此实验结果提示:在冬凌草甲素作用于L929细胞后激活了其他的不同于casPas。家族的蛋白作用于PARp。此外，冬凌草甲素通过增加Bax忍cl一2的表达比率，上调ERK蛋白磷酸化，抑制p38蛋白磷酸化而促使细胞死亡。 在HeLa细胞实验中，形态学变化及DNA电泳都证明冬凌草甲素诱导HeLa细胞凋亡，且启动了经典的信号途径，实验表明casPase一3抑制剂能阻止冬凌草甲素诱导的细胞死亡率，抑制caPase一3的活力升高，且减弱DNA片断化的形成。进一步的实验结果提示:冬凌草甲素通过改变Bax忍。1一xL与Bax/B。1一2的表达比率激活casPase一3，使其底物ICAD被降解，释放出激活casPase一3的核酶(cAD)引起细胞凋亡。另外，在冬凌草甲素的作用下，MAPK家族成员ERK与泪呵K被激活，不影响p38激酶的活化。通常，I‘Ba与核内因子哪一‘B结合，抑制其进入核内。实验中发现:IKBa的表达减少，磷酸化IKBa的表达增加，提示NF一KB被激活。有趣的是冬凌草甲素促使COX一2的表达，这一实验结果暗示:在HeLa细胞中COX一2的表达与癌症的发生无关。 本文在证明冬凌草甲素选择性杀伤癌细胞，对人外周血单核细胞具有很弱的细胞毒作用，并首次探讨冬凌草甲素在A375一s2细胞，L929细胞和HeLa细胞中诱导细胞死亡的机制。更多还原

【Abstract】 This dissertation reports that oridonin isolated from Rabdosia rubescens, induced cell death through different mechanisms in A375-S2, HeLa, and L929 cells.The studies demonstrate that oridonin significantly inhibited proliferation of several lines of tumor cells, including human malignent melanoma A375-S2 (IC50: 15.1 1.2 mol/L), chronic myelogenous leukemic cell K562 (IC50: 16.2 1.2 mol/L), human histocytic lymphoma U937 (IC50: 27.1 2.3 mol/L), promyelocytic cell HL-60 (IC50: 20.4 3.6 mol/L), and mouse fibrosarcoma L929 (IC50: 35.6 4.2 mol/L). Moreover, treatment with oridonin 34.3 mol/L for 12 h significantly inhibited A375-S2 cell growth, and showed minor cytotoxicity against PBMC, suggesting that oridonin showed selective antitumor activity and had less side effects on human normal cells.In oridonin-treated A375-S2 cells for 12 h, cysteine aspartic specific protease: caspase-3, -8 were activated at early stages, apoptotic bodies were formed, nuclear damage was observed by Hoechst 33258 staining and DNA fragmentation was exhibited. Oridonin increased the expression of the apoptosis inducer, Bax, decreased the expression of the anti-apoptotic protein, Bcl-xL, promoted the release of cytochrome c without affecting Bcl-2 expression, and activated down-stream caspase-9 in mitochondrial pathway. These observations indicated that appropriate dose of oridonin envisioned an initial premitochondrial phase that involved the Bcl-2 family of proapoptotic protein Bax that required the participation of caspase-9 and caspase-3. However, treatment with oridonin 137.4 mol/L for 12 h, the majority of A375-S2 cell underwent necrosis measured by LDH activity-based assay. Twelve hours after treatment with 34.3 mol/L oridonin, the ratio of Bax/ Bcl-xL protein expression was increased and release of cytochrome c were decreased by extracellular signal-regulated kinase (ERK) MARK inhibitor (PD98059) and phosphoinositide 3-kinases (PI3-K) inhibitor, wortmannin. Mitochondrial permeability transition (MPT) inhibitor, decylubiquinone, suppressed the release of cytochrome c without affectingBax expression. The activation of tumor suppression factor p53 by oridonin was also blocked by wortmannin. Taken together, Oridonin induced A375-S2 cell apoptosis by activating parallel p53, ERK and p38 pathways, increasing the ratio of Bax/Bcl-xL protein expression, and promoting the release of cytochrome c into cytosol, resulting in apoptotic cell death.The growth-inhibitory activity of oridonin against L929 cells is in time- and dose-dependent manner. Treatment with various concentrations of oridonin for 12 h, the majority of L929 cells underwent apoptosis as measured by LDH activity-based assay. Although apoptotic bodies were observed in oridonin-treated L929 cells, DNA fragmentation as a hallmark of apoptosis was not found. Pan-caspase inhibitor, z-VAD and caspase-3 inhibitor, z-DEVD sensitized L929 cells to oridonin instead of inhibition of apoptosis, however, PARP inhibitor (DPQ) effectively blocked oridonin-induced cell death. At 12 h treatment, PARP proenzyme was significantly cleaved. This result indicated that oridonin-induced L929 cell death required PARP degradation in caspases-independent manner. In addition, MEK/ERK inhibitor (PD98059) markedly blocked oridonin-induced cell death, whereas p38 inhibitor (SB203580) and JNK inhibitor (SP600125) weakly protected the cells against death. Treatment with 41.2 fonol/L oridonin for 12 h significantly induced a persistent ERK activation and p38 inactivation in L929 cells without evident changes in the protein levels. Moreover, oridonin increased the ratio of Bax/Bcl-2 protein expression, whereas it had no effect on the expression of Bcl-xL. These results indicated that regulation of Bcl-2 and MAPK families may be the effector mechanisms of oridonin-induced L929 cell death, independent of caspase pathway.In HeLa cells, oridonin induced oligonucleosomal fragmentation of DNA and increased caspase-3 activity, on the other hand, reduced the expression of inhibitor of caspase-3-a更多还原