【作者】 丁岩；

【导师】 陈晓光；

【作者基本信息】 中国协和医科大学 ， 药理学， 2005， 博士

【摘要】 细胞内蛋白激酶是控制细胞增殖、分化或凋亡的重要调控因子之一。蛋白激酶可以将细胞表面信号传导入细胞核内，引起基因表达改变。在已知的信号传导通路中，MAPK（mitogen-activated protein kinase，丝裂原激活的蛋白激酶）信号转导通路是近年来研究的一个热点。MAPK通路原件进化上高度保守，每个组件均由三个胞浆激酶构成：MAPKKK（mitogen-activated protein kinase kinase kinase，丝裂原激活的蛋白激酶的激酶的激酶），MAPKK（mitogen-activated protein kinase kinase，丝裂原激活的蛋白激酶的激酶），MAPK。目前普遍被接受的MAPK信号转导通路的模式途径是：Raf-MEK1／2-ERK通路，MEKK1-MEK4／7-JNK通路和TAK-MEK3／6-P38通路。人MEKK1蛋白的分子量为196kDa，由1495个氨基酸组成，是一个重要的MAPKKK蛋白激酶。本论文通过将人MEKK1基因转染入小鼠黑色素瘤细胞B16，经过筛选鉴定，建立了稳定高表达MEKK1的小鼠黑色素瘤细胞株M1B16。研究发现MEKK1基因可以抑制B16细胞的生长。与对照细胞相比，M1B16细胞在体外软琼脂集落形成的能力降低，体内在C57／BL6小鼠致瘤性降低，在Balb／c裸鼠肿瘤生长抑制。MEKK1基因使B16细胞形态变长，体内外生长均呈现类成纤维细胞形态；MEKK1可以使B16细胞粘附能力减弱，细胞内Calpain-1酶活性降低；使B16细胞体外侵袭运动能力增强，体内转移活性增加。这与多数关于MEKK1基因功能的报道一致。采用MTT法，就常见抗肿瘤药物对M1B16的作用进行了初步观察，结果表明与对照细胞B16和PB16相比，Taxotere和Taxol对M1B16的IC₅₀增加了约10倍，PD153035的IC₅₀提高了约20倍。这一结果提示本高表达细胞模型可能用于筛选针对微管和EGFR-MEKK1-MEK4／7-JNK通路的特异性抑制剂。总之，本文成功地建立了稳定高表达MEKK1的小鼠黑色素瘤细胞株M1B16，研究表明该细胞株可以作为细胞水平的药物筛选模型用于筛选针对MEKK1特异性的抑制剂。MEKK1作为三激酶通路的上游激酶在细胞的生长、增殖、运动、迁移以及机体的应激过程中发挥重要作用，但是国内外尚未见有对肿瘤细胞中MEKK1表达水平的报道。为了快速测定细胞中MEKK1的表达，本研究建立了一套直接竞争ELISA方法，可以快速测定细胞样品中MEKK1的表达水平，其灵敏度为0.17ng／mL，检测范围为0.1-10000ng／mL。方法学实验表明本方法稳定可靠，变异系数的范围在2.81到10.12之间，回收率在89.23％与109.32％之间。本方法在标准样品浓度在1.7×10^-4和10μg／mL之间时线性关系良好，相关系数R²为0.9937。总之，所有检测数据显示本方法重复性好、灵敏度高，是一种定量检测细胞样品中MEKK1表达水平的可靠方法。可以用于研究细胞中MEKK1的表达，快速筛选作用于MEKK1的药物。利用已建立的ELISA方法对常见细胞株中MEKK1的表达水平进行了检测。结果表明，人胰腺癌细胞（PC-3）和人胚胎脐静脉内皮细胞（ECV304）中MEKK1的表达明显高于其它细胞株，从另一个角度说明MEKK1在调节胰腺癌细胞生长、分化、增殖以及血管生成方面的重要作用。本实验室对从传统有毒草药萝摩科娃儿藤和三分丹中分离到的十余种抗肿瘤成分进行了大量的初筛和复筛，从中发现CAT在体内外均具有较强的抗肿瘤活性，且具有良好的量效关系。本文深入探讨了化合物CAT的体外抗肿瘤作用及其作用机制。MTT法发现CAT可抑制多种体外培养的肿瘤细胞的生长，在体外的半数抑制浓度IC₅₀范围在0.044～0.286μmol／L。SRB法观察了CAT对这些肿瘤细胞生长的影响，结果表明CAT可不同程度地抑制这些细胞的生长，半数抑制浓度GI₅₀范围在0.023～0.103μmol／L，完全抑制浓度TGI范围在0.079～0.390μmol／L。细胞生长曲线和集落形成实验表明，CAT可剂量依赖性地抑制人肝癌细胞Bel7402和人结肠癌细胞HCT-8的生长和集落形成能力。流式细胞分析表明，CAT使Bel7402细胞阻断于Gl和S期。Western Blot分析表明CAT处理Bel7402细胞12h，可使P53蛋白表达量增加，处理Bel7402细胞24h可使P16、P21和CyclinA蛋白的表达量明显升高。将CAT与DNA Topo I及pBR322 DNA在体外共同孵育可观察到对酶活性的抑制作用，结果表明CAT可明显抑制DNA Topo I的解旋活性，使超螺旋型DNA的量明显增加，同时加入CAT的DNA条带位移明显滞后。这一结果提示，CAT可能通过嵌入DNA，抑制DNA解聚及断裂DNA重新连接而发挥作用。CAT作用小鼠黑色素瘤B16-BL6细胞15h，使B16-BL6细胞侵袭穿过重组基底膜的能力明显下降，0.02、0.04和0.08μmol／L CAT使B16-BL6细胞的侵袭能力分别降低57％、75％和86％（P＜0.01）。经不同浓度CAT作用2h后，B16-BL6细胞与基底膜成分的粘附受到不同程度的抑制，并呈一定的剂量依赖关系。用底物酶谱法观察CAT对人肉瘤细胞HT-1080分泌基质金属蛋白酶能力的影响，结果表明不同浓度CAT作用24h，可以剂量依赖性地抑制MMP-2的分泌。以上结果均表明CAT在体外可有效作用于肿瘤细胞侵袭转移的各环节，阻断侵袭转移的发生。CAT对人胚胎脐静脉血管内皮细胞ECV304的增殖具有明显的抑制作用，MTT试验测得其半数抑制浓度IC₅₀为0.088μmol／L，SRB试验测得其半数抑制浓度GI₅₀为0.10μmol／L，完全抑制浓度TGI为0.22μmol／L。采用fibronectin作趋化剂，观察了CAT对ECV304细胞迁移能力的影响。结果表明，0.02、0.04和0.08μmol／L CAT可明显抑制ECV304细胞的迁移，对ECV304细胞的趋化性运动能力的抑制率分别为55％、65％和82％（P＜0.01）。用底物酶谱法观察CAT对ECV304细胞分泌基质金属蛋白酶能力的影响，结果表明不同浓度CAT作用24h，可以剂量依赖性地抑制MMP-2和MMP-9的分泌。CAT在体外表现出较强的抑制血管生成的作用，明显抑制ECV304细胞在Matrigel基质上形成的管腔结构。0.02、0.04和0.08μmol／L CAT处理ECV304细胞24h，可使其在Matrigel基质上形成管腔结构的能力分别降低至对照组的81.8％、54.6％和16.1％。另外CAT还可抑制KDR及MMP-9基因的表达。CAT对血管生成的抑制作用可能是其体内抑制肿瘤生长和侵袭转移的一个重要原因。综上所述，CAT在体外可广泛抑制不同组织来源的肿瘤细胞的生长，其作用机理可能与CAT嵌入DNA，抑制DNA Topo I的解旋活性，使细胞内与细胞周期调控相关的蛋白P53、P21、P16和CyclinA表达增加，细胞停滞于Gl和S期有关。同时CAT在体外还表现出较强的抗转移和抗血管生成作用。因此，CAT很可能成为一个新型具有自主知识产权，作用机制独特的潜在抗肿瘤药物。更多还原

【Abstract】 MEKKl is a 196-kDa serine-threonine kinase activated in response to a variety of stimuli, including EGF, lysophosphatidic acid, osmotic stress, UV light, and microtubule toxins. In this study, a subline of B16 mouse melanoma cells harboring MEKKl genes （M1B16） was established, and the expression of MEKKl could decrease the colony formation and tumor formation abilities of mouse melanoma B16 cells. The adherence ability and the Calpain-1 activity were also decreased in M1B16 cells referring to control cell lines. The motility and invasive ability were enhanced by the expression of MEKKl in vitro and in vivo. The morphology of B16 melanoma cells harboring MEKKl showed the dendrite outgrowth. All of these data indicate that M1B16 may be a suitable cell model to screen MEKK1 inhibitor for cancer metastasis therapy. MEKK1 is an important kinase in MAPK signaling pathway. Upon activating, MEKK1 participates in the regulation of the JNK and ERK pathways and is involved in the activation of NF-κB. In addition, MEKK1 is activated in response to changes in cell shape and the microtubule cytoskeleton. MEKK1 senses microtubule integrity, protects cells from committing to apoptosis, and contributes to the migration of fibroblasts and epithelial cells. In this study, a direct competitive ELISA to quantify total MEKK1 in human cell lines was developed. The procedure showed a high sensitivity （detection limit: 0.17 ng/mL）, good precision （coefficient of variation≤10.12） and acceptable linearity with a large range of MEKK1 concentrations （0.1-10000 ng/mL）. In a pilot study, this assay was used to quantify MEKK1 in different cell lines. In cancer cells, the range of MEKK1 is 0.02-85 ng/mg protein and its concentration was higher in pancreas cancer and umbilical vein cells than that in others. And this high expression of MEKK1 in these two cell lines indicates that MEKK1 may be a potential target in interfering with pancreas cancer and angiogenesis. Phenanthroindolizidine alkaloids are found primarily in the genera Cynanchum, Pergularia, and Tylophora of the Asclepiadaceae, but have also been reported from Hypoestes verticillaris （Acanthaceae）, Cryptocarya phyllostemmon （Lauraceae）, Ficus hispida and F. septica （Moraceae）. It is a small group of alkaloids known for its profound cytotoxic activity. It has been shown that the toxicity of phenanthroindolizidine alkaloids is due to inhibition of protein and nucleic acid synthesis. In this paper, the anti-tumor activities of a new phenanthroindolizidine alkaloid （CAT） against cancer cells in vitro were investigated to provide the molecular mechanism for CAT in the treatment of hepatoma.Effects of CAT on the growth of different cancer cell lines were assayed by MTT and SRB assay. MTT test showed that the IC₅₀ of CAT to cancer cells were in the range of 0.044～0.286μmol/L. Human hepatoma cell （Bel7402） was the most sensitive to CAT with the IC₅₀ of 0.044μmol/L. GI₅₀ evaluated by SRB assay was 0.023～0.103μmol/L. TGI evaluated by SRB assay was 0.079～0.390μmol/L. The inhibition of CAT on growth of Bel7402 and HCT-8 cells was in a dose-dependent manner, and the colony formation ability of Bel7402 and HCT-8 cells was inhibited by CAT （P＜0.05） significantly, too. To investigate the nature of growth inhibition caused by CAT, flow cytometric analysis was performed. The cells treated with increasing concentrations of CAT showed progressive accumulations in the G1 phase and S phase of the cell cycle. Topoisomerase I activity was measured by DNA relaxation assay. CAT inhibited Topoisomerase I activity in a dose-dependent manner. Western Blotting analysis was performed to observe the expression of P53, P21, P16, and CyclinA proteins. P53, P21, P16 and CyclinA protein expression were increased in a dose-dependent manner.After treated with CAT for 2h, the adhesive ability of Bl6-BL6 cells with basement membarane components （Matrigel） was markedly reduced in a dose-dependent manner. 0.02, 0.04 and 0.08μmol/L CAT inhibited significantly the migration of Bl6-BL6 cells with the inhibition rate of 57％, 75％and 86％（P＜0.001）, respectively. Secretion and activation of MMP-2 in serum-free culture medium of HT-1080 cells treated by CAT for 24h were suppressed by a dose-dependent mode. These results implied that CAT decreased degradation of basement membrane and invasive potentials by inhibiting the adhesion, migration and invasion ability of tumor cells and suppressing the secretion and activation of MMP-2.The anti-proliferation effect of CAT on human umbilical vein endothelial cell （ECV304） was demonstrated by SRB assay with GI₅₀ of 0.10μmol/L and with TGI of 0.22μmol/L. MTT test showed that the IC₅₀ of CAT to ECV304 was 0.088μmol/L. The chemotaxis mobility of ECV304 cells induced by fibronectin （FN） was significantly restrained by CAT at the different concentrations, the inhibitory rate of 0.02, 0.04 and 0.08μmol/L CAT on migration potential was 55％, 65％and 82％, respectively. Three-dimension in vitro angiogenesis system showed that CAT inhibited tube formation of ECV304 cells seeded on Matrigel, the multicellular capillary-like structure formation was suppressed to 81.8％, 54.6％and 16.1％of untreated control by 0.02, 0.04 and 0.08μmol/L CAT, respectively. With RT-PCR analysis, KDR and MMP-9 gene expression of ECV304 cells were inhibited by CAT treatment. CAT inhibited angiogenesis by suppressing endothelial cells proliferation, migration and KDR gene expression.In summary, it was found that CAT inhibited growth of various cancer cells in vitro, the metastasis of Bl6-BL6 cells in vitro and the angiogenesis process. CAT could induce progressive accumulations of Bel7402 cells at the Gl phase and S phase. This blockade was related to the Topoisomerase I inhibition by CAT. The changes of P53, P21, P16, and CyclinA protein expression levels may play a major role in the cell cycle arrest triggered by CAT. Inhibition of CAT on MMP secretion may play a key role in suppression both on tumor growth and on metastasis. The effects of CAT on angiogenesis were associated with inhibition of expression of KDR genes. The results reported herein may suggest potential clinical application of CAT against cancer, especially for those refractory to current chemotherapy.更多还原