【作者】 韩炯；

【导师】 俞强； 药立波；

【作者基本信息】 第四军医大学 ， 生物化学与分子生物学， 2002， 博士

【摘要】 脱落凋亡(anoikis)是凋亡的一种形式，它是由于正常的上皮细胞或内皮细胞脱离与ECM的联系而引起的。其生物学意义在于防止这些脱落的细胞种植于其他不适当的地方继续生长。大多数来源于上皮或内皮组织的肿瘤细胞，尤其是容易发生转移的恶性肿瘤细胞，则失去了这种特性，由于瘤细胞从瘤体上脱落后并不发生凋亡，因而可以种植到其他部位继续生长。这种存在于某些肿瘤细胞中的对脱落凋亡的敏感性降低被认为是肿瘤发生转移的一个重要机制。转移瘤的发生是临床上肿瘤病人死亡的一个重要原因，而且这种现象极其多见，例如乳腺癌、肺癌、肝癌、胃癌、前列腺癌等恶性肿瘤都很容易发生转移。因此，阐明肿瘤细胞抗脱落凋亡现象的信号转导过程将会是一项非常有前景的研究课题。一方面对于理解参与脱落凋亡和抗脱落凋亡过程的信号分子和信号转导途径具有重要的理论意义，另一方面对于发展抑制肿瘤转移新策略同样具有重要的应用价值。 为深入理解肿瘤细胞抗脱落凋亡的分子机制，我们首先通过DNA ladderring检测、FCM检测以及软琼脂集落形成实验筛选了一些乳腺癌细胞系，包括Bcap-37、MCF-7、MDA-NB-231以及SK-BR-3，观察它们对脱落凋亡的敏感性。结果显示，来源于狗正常肾脏上皮的阳性对照MDCK细胞对脱落凋亡是敏感的，表现为琼脂糖凝胶电泳检测到凋 第四军医大学博士学位论文 第6页亡特征性的片段化DNA，细胞周期中高比例的亚二倍体峰的出现，以及在软琼脂中不能形成集落。而Bcap－37、MCF－7、MDA－n－231三种乳腺癌细胞则具有抗脱落凋亡的特性，分别与它们各自贴壁培养的细胞比较，胞浆DNA琼脂糖凝胶电泳表现无明显差异，FCM所检测到的凋亡峰也无明显区别。而且，这三种乳腺癌细胞在软琼脂中都具有形成集落的能力。SK．BR－3细胞同样是抗脱落凋亡的；但它却不能在软琼脂中形成集落，也许与ECM联系的失去对SK－BR－3细胞的悬浮生长有一定的影响。 对于筛选出的三种抗脱落凋亡的肿瘤细胞系，它们的抗脱落凋亡特性究竟与哪个或哪些信号分子有关呢？首先，我们观察了一些信号分子的特异抑制剂对肿瘤细胞抗脱落凋亡特性的影响，期望通过对脱落凋亡的抑制来提示我们与脱落凋亡相关的信号分子。其中包括：HERZ的特异抑制剂AG825；，PDGFR的特异抑制剂AG17；p38MAPK的特异抑制剂sB203580：＊D－K的特异抑制剂LY 29以m2；MAP KK（K）的特异抑制剂PD98059。胞浆DNA电泳显示在短时间内（15h），这些抑制剂未能诱导悬浮培养的肿瘤细胞出现凋亡现象，但在软琼脂集落形成实验中，每种抑制剂对肿瘤细胞在软琼脂中的生长则具有不同的影响。AG17对三种乳腺癌细胞均有明显的抑制作用，PD98059可以部分抑制MDA－MB－231的集落形成。其余的抑制剂则没有明确的生长抑制现象。这些结果提示我们，PDGFR信号通路可能与三种乳腺癌细胞的抗脱落凋亡特性相关。另外对于 MDA．MB．231细胞来说，MAPK信号转导通路在其抗脱落凋亡过程中也许起着一定的作用。由于信号转导网络系统的复杂性，这两种信号转导通路究竟扮演着什么样的角色尚待进一步的研究。 在抑制剂分析实验中，作用比较明确的是广谱蛋白酪氨酸激酶抑制剂genistein，尽管短期（24小时内）作用不是很明显，胞浆DNA琼脂糖凝胶电泳以及FCM均未能检测到它对悬浮培养的三种乳腺癌细胞的诱导凋亡作用。但它可以完全抑制它们在软琼脂中的生长，经 western blotting检测，在 genistein的作用下细胞中总体蛋白酪氨酸的磷酸化水平也有不同程度的下降。这提示我们肿瘤细胞的抗脱落凋亡特性很可能仍然是由蛋白酪氨酸激酶来介导的。 我们进一步检测了几种相关信号通路中的关键信号分子在贴壁培养与悬浮培养时的表达水平及磷酸化状态的改变。发现两种重要的参与细胞存活信号通路的信号分子ERKI／2以及AKT（分别参与MAPK信号通路以及PD－K信号通路）的表达水平在 Doann’Uat ofBtochemtw and ilecUler Btotort FMMU 第四军医大学博士学位论文 第7页贴壁细胞与悬浮细胞中没有明显的差异。但是未能检测到它们磷酸化水平的差异，因而它们是否参与所检测的三种乳腺癌细胞抗脱落凋亡的过程尚不能明确。FAK的磷酸化水平在悬浮培养的细胞中是下降的，这与文献报导一致。衔接分子 PI 30CAS在悬浮培养的乳腺癌细胞中的表达水平与贴壁培养的细胞比较有不同程度的增加，尤其是在MDA－MB－23细胞中，随着悬浮培养的时间延长，这种趋势更为明显。 我们同时检测了三种乳腺癌细胞在悬浮培养时总体蛋白酪氨酸磷酸化水平的改变。大多数蛋白酪氨酸磷酸化的水平在悬浮培养的乳腺癌细胞中是下降的。例外的是在悬浮培养的BCap－37细胞以及MDA－MB－231细胞中，在66二kD与97．4kD之间有一条带，其磷酸化水平与贴壁培养的细胞相比明显增高。此外，在 MDA－MB－23细胞中，有一大分子量（100kD以上）的分子的酪氨酸磷酸化水平在悬浮培养时明显?更多还原

【Abstract】 Anoikis is a form of apoptosis triggered by disruption of cell-matrix contacts. It is often seen in normal epithelial and endothelial cells. The significance of anoikis is that it may prevent the accidentally detached cells from reattaching to new matrices and growing dysplastically. On the other hand, most tumor cells derived from epithelial tissues have lost this characteristic, especially those malignant tumors that are easy to metastasize. The cells detached from tumors do not undergo apoptosis and can colonize elsewhere and grow. It is an important mechanism of tumor’s metastasis, which attributes to clinical death of most of tumor patients and is often seen in breast, lung, hepatic, gastric and prostate tumors. Therefore, it is important to elucidate the molecular mechanism of tumor cells’ anoikis resistance. It will help us to understand the molecules and pathways related to this phenomenon and it has a practical value for developing new strategies to prevent tumor metastasis.To understand the molecular mechanism of tumor cell resistance of anoikis, we first analyzed several breast cancer cell lines, including Bcap-37, MCF-7, MDA-MB-231 and SK-BR-3, to determine their sensitivity to anoikis through DNA laddering assay, FCM and soft agar assay. The results showed that the MDCK cells, which are derived from normal canine kidney, are sensitive to anoikis. Fragmented DNA could be detected by agarose gelDepartment of Biochemistry and Molecular Biology, FMMU10electrophoresis. High proportion of cells was in Sub-Gl phase of eel] cycle and the cells could not grow in soft agar to form colonies. On the contrary, the three breast cancer cell lines, Bcap-37, MCF-7 and MDA-MB-231, are anoiMs resistant, as indicated by DNA laddering and FCM assays. Furthermore, they all grow in soft agar and form colonies. SK-BR-3 cells are also anoikis resistant, but it could not form colonies in soft agar. Therefore, detachment may have some effects on the growth of SK-BR-3 cell cultured in suspension.Next we tried to find out which signaling molecules in the selected three tumor cell lines are related to their resistance to anoikis. We first investigated the effects of specific inhibitors of several signaling molecules on the tumor cell resistance of anoikis. These are KER2 inhibitor AG825; PDGFR inhibitor AG17; P38MAPK inhibitor SB203580; PD-K inhibitor LY294002 and MAPKK (MEK) inhibitor PD98059. Agrose gel electrophoresis showed that these inhibitors failed to induce suspended tumor cells to undergo apoptosis in a short time (15h). But they had different effects on tumor cells grew in soft agar. AG17 could inhibit the growth of three tumor cell Lines apparently and PD98059 could inhibit MDA-MB-231 cell forming colonies partially. The rest had no apparent effect on cell growth. These results imply that PDGFR may be involved in anchorage-independent growth of the tumor cells. MAPK signaling pathway may play a role hi the process of MDA-MB-231 cell’s resistance to anoikis. Because of the complexity of signal transduction networks, the importance of these two signaling pathways in tumor cells’ resistance to anoikis are need to be further studied.The effect of the general protein tyrosine kinase inhibitor genistein on tumor cell anoikis resistance was also analyzed. Although the effect of genistein on anoikis was not apparent in a short time (less than 24h), as indicated by both agrose gel electrophoresis assay and FCM assay, genistein could inhibit the growth of the tumor cells in soft agar completely. The total levels of protein tyrosine phosphorylation in the three tumor cells upon genistein treatment were also decreased to some extent. These data imply that the tumor cell resistance to anoikis is mediated by protein tyrosine kinases.Department of Biochemistry and Molecular Biology, FMMUFurthermore, we analyzed the changes in expression and phosphorylation of key signaling molecules in several related signal transduction pathways. We found that two important survival signaling molecules, ERK1/2 and AKT, h更多还原