【作者】 史颖弘；

【导师】 樊嘉；

【作者基本信息】 复旦大学 ， 外科学， 2005， 博士

【摘要】 肝细胞肝癌是常见的恶性肿瘤之一,随着诊断技术及治疗手段的发展,近二十年来疗效已经有了很大的提高,但肝癌的转移复发仍是影响长期疗效的重要因素。深入研究肝癌转移的分子机制有助于发展更有效的治疗手段。已知转移灶的形成涉及肿瘤细胞的脱落、粘附、出血管、肿瘤血管生成及微转移灶的生长等,是一多步骤、低效率的过程。血流的冲击、低氧、营养缺乏、免疫系统的攻击、不适的组织微环境等将诱导大部分细胞死亡,转移过程细胞对死亡刺激的反应直接影响转移灶形成的成功率。高转移潜能细胞可能获得了某种耐受细胞死亡的机制而易于形成转移灶或表现为对药物治疗的抵抗,因此研究肿瘤细胞死亡与转移相互作用的分子机制有很重要的意义。 细胞死亡可以分为三大类,凋亡(Ⅰ型)、自噬性细胞死亡(Ⅱ型)、Ⅲ型(坏死)。凋亡及自噬性细胞死亡,甚至坏死均被认为是程序化细胞死亡方式,受严格的调控,有一系列基因及信号传导通路参与细胞死亡的调节。其中凋亡在肿瘤的发生、分化、耐药等环节中所起的重要作用已得到大量研究证实,但肿瘤细胞凋亡及自噬性细胞死亡在肿瘤转移中的作用尚不是很明确。 Caspases的激活障碍或过度的抑制是细胞耐受凋亡的重要机制。凋亡蛋白抑制物(IAPs)是唯一已知的内源性凋亡caspases抑制物,已知人类中有cIAP1、cIAP2、NAIP、Survivin、XIAP、Bruce、ILP-2及Livin八种IAP蛋白,其中许多蛋白发现在肿瘤中过度表达。但IAPs在肝癌中的表达及IAPs的表达变化对细胞凋亡、肝癌转移潜能的影响还有待阐明。我们研究发现高转移肝癌耐受不同凋亡刺激诱导的凋亡,而且可能与XIAP的过度表达有关。在不同转移潜能的肝癌细胞株中,上调/下调XIAP的表达可以改变细胞的凋亡敏感性,模拟Smac的化合物Compound 3抑制XIAP能增加细胞的凋亡敏感性。体外实验中,XIAP的调节能改变肝癌细胞的转移侵袭潜能。 自噬(Autophagy)在多细胞生物中主要生理功能为蛋白质的降解及细胞器的转换,维持细胞自身的稳定。但在持续或过度的刺激下有可能导致自噬性细胞死亡。自噬不但是细胞正常的生理反应而且参与很多病理过程,但在肿瘤中的作用还有争议。细胞在氨基酸缺乏时将诱导自噬,可能是细胞的一种保护机制。我们发现高转移潜能的肝癌细胞株耐受氨基酸缺乏诱导的细胞死亡,而且参与自噬过程的自噬相关基因ATG7、ATG8明显低表达,提示高转移潜能肝癌可能比较耐受自噬性细胞死亡,自噬性细胞死亡在肝癌转移中可能有重要作用。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most common tumors worldwide. The prognosis of HCC has been obviously improved in recent two decades with the early diagnosis and effective treatments, but the tumor recurrence and metastasis are still the major obstacles for the long term survival. To elucidate the molecular mechanism of HCC metastasis will contribute to develop more effective treatment methods. The process of metastasis can be briefly summarized to a series of sequential steps that include intravasation, transport via the circulatory system, arrest at a secondary site, extravasation, and growth in a secondary site. However, metastasis is an inefficient process by which only a few of the cells that leave a primary tumor site survive to form the metastatic foci. It is likely that metastasis can fail at many of the stages of the process due to cell destruction by the host immune system, blood flow turbulence, hypoxia, nutrition deprivation, and unsuitable tissue microenvironments. The death of tumor cells in the processes of metastasis will influence the metastatic efficiency directly. Highly metastatic tumor cells have more chances to survive and/or be resistant to chemotherapy by some mechanisms to conquer the cell death, so it is important to explore the molecular mechanism and the relationship between the cell death and metastasis.Cell death can be divided into apoptosis (type I), autophagic cell death (type II), and necrosis (type III). Apoptosis and autophagic cell death, even necrosis, have been defined as programmed cell death and can be regulated by different molecular pathways. The function of apoptosis is well known that in carcinogenesis, cell differentiation, and chemotherapy resistance. But the role of apoptosis and autophagic cell death in the metastasis of hepatocellular carcinoma still need to be elucidated.Failure to activate caspases or excessive suppression of caspases can cause resistance to the apoptosis. The inhibitors of apoptosis proteins (IAP) represent the only known family of endogenous caspases inhibitors. To date, eight human IAPs have been identified: c-IAP1, C-IAP2, NAIP, Survivin, XIAP, Bruce, ILP-2, and Livin, several of which are overexpressed in different cancers. However, little is known about the expression of IAPs in hepatocellular carcinoma and the relationship with HCC metastasis. We identified that a hepatocellualr carcinoma cell line with highlymetastatic potentials is more resistant to apoptosis induced by different apoptotic stimuli which can be due to overexpression of XIAP. Overexpressing or knocking down the level of XIAP in hepatocellular carcinoma cell line with different metastatic potentials can alter the sensitivity to apoptosis, and the Smac mimic, Compound 3, can potentiate the TNF- a induced cell death in thesis HCC cell lines. Furthermore, Modification of the level of XIAP can alter the HCC metastatic potentials in vitro.Autophagy is an evolutionarily conserved mechanism of protein and organelle degradation which has been observed in organisms as diverse as yeast and mammals. Autophagy is a normal physiological process to maintain homeostasis and enhance survival. Autophagic cell death may occur if the process is carried beyond a threshold. But it is controversial whether autophagy is a cell survival or cell death mechanism especially in human tumors. It has been described that autophagy is induced after amino acid deprivation as a protective mechanism, attempting to recycle the materials during starvation. We have found the HCC cell line HCCLM3 with highly metastatic potential is more resistant to amino acid starvation induced cell death and expresses relatively low expression level of ATG7 and ATG8, which are required for the autophagy process, These results may indicate that autophagic cell death can be regulated in HCC metastasis and HCC with highly metastatic potentials are more resistant to autophagic cell death.MethodsCell viability and apoptosis were determined by MTT assay, trypan blue exclusion, and nuclear staining with hoechest 33342. Caspases activation was assessed by the cleavage of site-selected tetrapeptide reportor substrates with the relative specificity for caspases 8(LETD-AFC), caspase 9(IETD-AFC), or caspase 3(DEVD-AFC). The expression levels of Bcl-2 family proteins, IAP family proteins, ATG family proteins, and other proteins in the three HCC cell lines were assessed by immunoblotting. XIAP was overexpressed by adenovirus Ad-Y5-XIAP and knocked down by pSIREN-shRNA-XIAP expression vector or SiRNA-XIAP RNA duplex. In vitro metastatic capability was evaluated by anchorage-independent growth assay and matrigel invasion assay.Results1. HCC cell line HCCLM3 with highly metastatic potentials is resistant to apoptosis induced by different apoptotic stimuli.Compared to the two low metastatic potential cell lines, SMMC-7721 and MHCC97L, HCCLM3 with highly metastatic potentials is more resistant to different apoptotic stimuli such as TNF-a , Etoposide, STS, MG132, and H2O2, which can induced apoptosis through either extrinsic or intrinsic pathway. The activation of caspases in HCCLM3 is milder than in SMMC-7721 and in MHCC97L cells. The broad spectrum caspase inhibitor z-VAD-fmk can partially reverse the apoptosis induce by TNF- a and Etoposide.2. The resistance of apoptosis in HCCLM3 cell is due to overexpression of XIAPIn order to find the key molecule contributing to the anti-apoptotic capacity in the highly metastatic HCC cell line, we examined the expressions of the Bcl-2 family proteins, IAP family proteins, and other apoptosis related proteins. Markedly increased expression of XIAP in HCCLM3 cell was found, which correlated with its metastatic potentials.3. Modification the expression of XIAP alter the sensitivity to apoptosisA Smac mimic small molecule Compound 3 can potentiate the TNF- a induced apoptosis in all three cell lines by counteracting the inhibitory effects of XIAP. In SMMC-7721 cell with low level XIAP expression, overexpression of XIAP with adenovirus Ad-Y5-XIAP can decrease the sensitivity to etoposide, t^C^and MG132 treatments. In HCCLM3 cell with markedly increased expression of XIAP, knocking down the XIAP level with siRNA-XIAP can sensitize the apoptosis to etoposide and STS.4. Modification of the expression of XIAP can change the metastatic potential in vitroIn SMMC-7721 cells with a low level of XIAP expression, overexpression of XIAP with adenovirus Ad-Y5-XIAP can increase the metastatic potentials in vitro as analyzed by the anchorage independent growth assay and the matrigel invasion assay. In HCCLM3 cell with markedly increased expression of XIAP, knocking down the XIAP level with siRNA-XIAP can decrease the metastatic potentials in vitro as analyzed by the anchorage independent growth assay and the matrigel invasion assay.5. The highly metastatic HCCLM3 is resistant to amino acid deprivation induced cell death.SMMC-7721 cell is very sensitive to amino acid starvation induced cell death and all the cells almost died within 48 hours, whereas the HCCLM3 is very resistant更多还原