【作者】 张莹；

【导师】 吴立军； 池岛乔；

【作者基本信息】 沈阳药科大学 ， 药物化学， 2003， 博士

【摘要】 本文对吴茱萸碱抗肿瘤活性进行了较为系统的研究，主要阐述了吴茱萸碱在三种肿瘤细胞：人黑色素瘤A375-S2细胞，人宫颈癌HeLa细胞，小鼠纤维肉瘤L929细胞中诱导细胞死亡的不同机制。 在测定吴茱萸碱对四种人源肿瘤细胞（黑色素瘤A375-S2细胞；宫颈癌HeLa细胞；急性单核白血病THP-1细胞；乳腺癌MCF-7细胞）和四种鼠源癌细胞（小鼠纤维肉瘤L929细胞；突变Buffalo大鼠肝癌BRL细胞；小鼠肉瘤S180细胞；小鼠宫颈癌U14细胞）细胞毒作用时发现四种人源癌细胞均对吴茱萸碱敏感，半数抑制浓度分别为：IC₅₀（A375-S2）：14.7±1.9 μmol·L^-1，IC₅₀（HeLa）：15.4±0.7μmol·L^-1，IC₅₀（THP-1）：18.1±1.1 μmol·L^-1，IC₅₀（MCF-7）：22.7±1.3 μmol·L^-1，但四种鼠源细胞中只有L929细胞对吴茱萸碱敏感，IC₅₀值为20.3±3.5 μmol·L^-1。与化疗药物放线菌素D，顺铂，及5-氟尿嘧啶对比，吴茱萸碱对人外周血单核细胞（PBMC）无细胞毒作用，这提示吴茱萸碱具有较强抗癌活性的同时毒副作用较小。 在A375-S2细胞中，Hoechst 33258荧光染色法证明吴茱萸碱作用细胞12h时出现明显凋亡小体，DNA电泳则表明吴茱萸碱作用24h时出现坏死特有的弥散泳带。采用LDH活力测定进一步证明吴茱萸碱处理细胞24h之内，细胞主要通过凋亡途径死亡，24h后坏死途径被启动。在吴茱萸碱诱导的早期凋亡途径中，caspase-3，8和9被激活，caspase家族抑制剂，caspase-3，8，9抑制剂均可显著抑制吴茱萸碱引起的凋亡，但这种作用在24h后即消失。应用Western blot法发现吴茱萸碱可明显上调促凋亡蛋白Bax的表达，而且同时下调抗凋亡蛋白Bc1-2的表达，这一切表明在早期吴茱萸碱诱导的凋亡是由caspase家族和Bc1-2家族共同调控的。吴茱萸碱作用细胞24 h后，p38抑制剂（SB203580）可部分抑制细胞死亡，与caspase家族抑制剂联用可增强这种抑制作用；与此相反ERK抑制剂（PD98059）可促进吴茱萸碱诱导的细胞坏死。我们进一步应用Western blot法证明吴茱萸碱虽不能影响p38蛋白表达但可上调磷酸化p38表达，即激活p38激酶；吴茱萸碱可减少ERK及磷酸化ERK蛋白表达。这些结果证明MAPK家族调控着吴荣英碱晚期启动的坏死途径。流式分析结果表明吴茉英碱对A3 75．SZ细胞周期无阻滞作用，细胞死亡发生于细胞周期各个阶段。 在HeLa细胞中，吴茉英碱启动了经典凋亡途径。荧光染色出现凋亡小体，DNA电泳呈现DNA梯状泳带，caspase家族抑制剂，c卿ase上，8，9抑制剂均可显著抑制吴茉英碱引起的凋亡。c卿ase活力测定证明caspase上，8，9活力升高，但caspase－8激活明显滞后于caspase－3和 caspase－9的激活。应用 Western blot法证明吴莱莫碱可上调Bax表达但下调Bcl－2的表达。采用LDH活力测定法亦证明吴莱莫碱在HeLa细胞中主要启动了凋亡途径而不象在A3 75S 细胞中还启动了坏死途径。虽然JNK抑制剂（SP600125）可协同促进吴茉英碱在HeLa细胞中的细胞毒作用，但吴荣美碱并不影响JN’K及磷酸化JN’K的表达。流式分析术结果表明吴某某碱可将HeLa细胞周期阻滞在GZ／M期发生大量凋亡。 在L929细胞中吴莱莫碱诱导非经典性凋亡，虽然荧光染色出现凋亡小体，且LDH活力测定表明吴某某碱引起的细胞死亡主要由凋亡途径进行，但DNA电泳并未出现DNA Ladder，而且最为意外的是c呷ase家族抑制剂及caspase上抑制剂可明显促进吴茉美碱引起的细胞死亡。流式分析结果表明低剂量5卜mol＊‘吴莱美碱可将细胞周期阻滞在 GZAI期，而高剂量 20 pmol·L“‘吴荣美碱可将细胞周期阻滞在GO／GI期。 本文在证明吴荣英碱对癌细胞杀伤作用具有选择性，且对人外周血单核细胞 （PBMC）无细胞毒作用的基础上，首次探讨了吴茉英碱在A375＊ 细胞，HeLa细胞和L929细胞中诱导细胞死亡机制，发现在三种细胞中吴某英碱发挥抗癌活性机制各不相同，这可以进一步解释吴荣英碱抗癌作用的靶向性及选择性。更多还原

【Abstract】 This dissertation mainly demonstrated that evodiamine induced cell death through different mechanisms in A375-S2, HeLa, and L929 cells.The studies found that evodiamine obviously inhibited proliferation of several lines of tumor cells, including human malignant melanoma A375-S2 (IC50: 14.7±1.9 mol.L-1), human cervical cancer HeLa (IC50: 15.4±0.7mol.L-1), human acute monocytic leukemic THP-1(IC50:18.1±1.1mol.L-1), and human breast adenocarcinoma MCF7 (IC50: 22.7±1.3 umol.L-0), mouse fibrosarcoma L929 (IC50: 20.3±3.5mol.L-1), but no cytotoxic effects were observed on mouse sarcoma 180 cell (S180), mouse uterocervical cancer U14 cell and Buffalo rat liver BRL cell. Moreover, evodiamine had less influence on human peripheral blood mononuclear cells (PBMC) growth, compared to actinomycin D, cisplatin and 5-Fu, suggesting that evodiamine showed selective anti-tumor activity and had less side effects on human normal cells.In evodiamine-treated A375-S2 cells for 12 h, Hoechst staining showed obvious apoptotic bodies. However, at 24 h, DNA electrophoresis appeared smear-like DNA fragmentation, which was a hallmark of necrosis. LDH activity assay demonstrated that before 24 h incubation with evodiamine, apoptosis pathway was mainly responsible for cell death in A375-S2 cells, but necrosis pathway was initiated after 24 h. At the early stage, evodiamine activated caspase-3, 8, and -9, moreover, pan-caspase inhibitor (z-VAD-fmk), caspase-3 inhibitor (z-DEVD-fmk), caspase-8 inhibitor (z-IETD-fmk), and caspase-9 inhibitor (z-LEHD-fmk) had apparently inhibitory effects on evodiamine-induced cell death, which disappeared after 24 h. Furthermore, it was revealed that evodiamine up-regulated Bax expression level but down-regulated Bcl-2’s with Western blot assay, suggesting that evodiamine-induced apoptosis was mainly mediated by both caspase family and Bcl-2 family. At the early stage, p38 inhibitor (SB203580) partially blocked evodiamine-induced cell death, which was augmented by combination SB203580 and z-VAD-fmk. Oppositely, ERK inhibitor (PD98059) enhanced evodiamine-induced cell death.Evodiamine did not alter p38 expression, but increased the phosphorylation of p38. However, evodiamine down-regulated both ERK expression and the phosphorylation of ERK. These suggested that MAPK family were responsible for evodiamine-induced necrosis at the late stage. Otherwise, evodiamine had no influence on cell cycle in A375-S2 cells, and evodiamine-induced cell death appeared at all of cell cycle phases.In HeLa cells, evodiamine initiated classic apoptotic pathway. Hoechst 33258 staining showed apoptotic bodies, and DNA electrophoresis appeared DNA ladder, which were believed to be the hallmark of classic apoptosis. Pan-caspase inhibitor and caspase-3, caspase-8, and caspase-9 inhibitors blocked evodiamine-induced apoptosis, moreover, caspase-3, 8, and caspase-9 were activated. However, the activation of caspase-8 was delayed, compared to caspase-3 and caspase-9. Bcl-2 expression was down-regulated and Bax expression was up-regulated by evodiamine. LDH activity assay also demonstrated that necrosis pathway did not involve in the evodiamine-induced HeLa cell death. Although INK inhibitor (SP600125) promoted the apoptosis, evodiamine failed to influence the expression of INK and JNK phosphorylation. Moreover, evodiamine induced cell cycle arrest at G2/M phase.It was demonstrated by LDH activity assay that evodiamine induced cell death through apoptosis pathway in L929 cells. Although apoptotic bodies were observed in evodiamine-treated L929 cells stained with Hoechst 33258, DNA fragmentation, a hallmark of apoptosis, was not detected. Unexpectedly, pan-caspase inhibitor (z-VAD-fink) rendered the cells even more sensitive to evodiamine and caspase-3 inhibitor (z-DEVD-fmk) also weakly augmented the cell death. Moreover, evodiamine caused G2/M arrest at the low dose, but GO/G1 arrest at the high dose.In summary, we first demonstrated that evodiamine induced cell death through distinct mechanisms in A375-S2 , HeLa, an更多还原