脂肪细胞因子衍生物对乳腺癌侵袭和转移的影响

【作者】 潘兵；

【导师】 韩佩珍； 卢圣栋； 张春明；

【作者基本信息】 中国协和医科大学 ， 影像医学与核医学， 2008， 博士

【摘要】 肥胖是一个在全球范围内持续增加的公共健康问题,它不仅与胰岛素抵抗和2型糖尿病密切相关,而且也是患一些常见肿瘤的风险因素,如乳腺癌、子宫内膜癌、结肠直肠癌以及前列腺癌等。肥胖导致一些内源性激素分泌的改变,包括类固醇激素、胰岛素和IGF系统,这些激素能影响细胞分化和血管再生。近年发现,脂肪组织不仅是能量储存的场所,还是一个非常活跃的内分泌代谢器官,分泌多种多肽物质,即脂肪细胞因子,包括VEGF、HGF、LEP、TNF-α、HB-EGF、IL-6、抵抗素以及脂联素等等,它们与肿瘤尤其是乳腺癌的发生发展密切相关,但作用机制还未完全阐明,绝大多数均停留在流行病学水平,实验证据较少。恶性肿瘤的侵袭和转移是一个复杂过程,其中降解细胞外基质是重要的一步。众多研究证实,MMPs与细胞外基质的降解密切相关。MMPs是一类具有锌离子依赖性的蛋白水解酶超家族,不同成员降解ECM中的不同成分。几乎所有的ECM成分均能被MMPs降解。TIMPs的功能主要是抑制MMPs的活性,其作用方式较为复杂,大多数情形下二者以1:1的比例结合成复合物,使MMPs活性丧失。众多研究表明,肿瘤中TIMPs的表达明显降低。鉴于目前关于脂肪细胞因子抵抗素抑制肿瘤细胞侵袭和转移作用的研究尚未见到报道,本文首次利用与人抵抗素具有同源性的抵抗素13肽,研究抵抗素对人乳腺癌细胞体内体外侵袭转移及相关机制的影响。本研究采用MTT法和平板克隆形成实验测定抵抗素13肽在5ng/ml～10μg/ml质量浓度范围内,按照时间依赖方式和剂量依赖方式抑制MDA-MB-231乳腺癌细胞的增殖和克隆形成,即随着药物作用时间的延长和药物剂量的增加,对肿瘤细胞增殖作用的抑制也增强。应用Transwell模型重建基底膜实验,发现抵抗素13肽在5ng/ml～10μg/ml质量浓度范围内能按照剂量依赖方式降低肿瘤细胞的侵袭能力。同时应用Transwell小室实验和划痕迁移实验,证实抵抗素13肽在5ng/ml～10μg/ml质量浓度范围内,也能按照剂量依赖方式降低肿瘤细胞的迁移运动能力。采用粘附实验发现抵抗素13肽在5ng/ml～10μg/ml质量浓度范围内的粘附能力也受到抑制。随后应用流式细胞仪测得抵抗素13肽在5ng/ml～10μg/ml质量浓度范围内,均能诱导肿瘤细胞凋亡,但是在其最高浓度10μg/ml作用下其凋亡率也低于10%,由此我们可以得出抵抗素13肽在一定程度通过诱导肿瘤细胞凋亡来降低肿瘤细胞的增殖、粘附、侵袭和迁移能力,但结果证明这不是最主要的途径。接下来,在本研究中应用明胶酶谱法和Western blotting法分析抵抗素13肽对肿瘤细胞分泌MMP-2和MMP-9及其组织抑制物TIMP-1和TIMP-2的影响,发现抵抗素13肽在50ng/ml及以上浓度时,MMP-2蛋白活性形式及酶原表达量以及MMP-9蛋白活性形式表达量随浓度的升高而下降,而它们的组织抑制物TIMP-1和TIMP-2的蛋白表达量则随浓度的升高而升高。由此我们可以推测抵抗素13肽对肿瘤细胞侵袭及粘附能力的降低可能是通过调节基质金属蛋白酶及抑制物的表达,保护基底膜不受破坏来实现的。最后,本研究还通过重建肿瘤模型进行抵抗素13肽体内抗癌作用研究,发现抵抗素13肽在体内也能抑制实体瘤的生长,除了肝脏有轻微的炎症外,并对其它的脏器并未产生毒理作用。解剖也没有看到预期中的卵巢囊肿现象的发生。另外,脂联素是作为目前发现的唯一抗血管生成的一种脂肪细胞因子,在流行病学以及临床调查研究发现,与乳腺癌的发生密切相关,在血清中的浓度与乳腺癌的发生呈负相关。本研究应用人脂联素重组体观测脂联素在乳腺癌的体外侵袭迁移作用,与流行病学调查结果一致。脂联素在体外能够抑制乳腺癌细胞的增殖、粘附、侵袭以及迁移能力,同时按照剂量依赖方式降低MMP-2和-9的活性及蛋白表达量的水平,以及按照同样方式增加TIMP-1和-2的蛋白表达水平。由此,推测脂联素重组体抑制肿瘤细胞侵袭过程可能是通过调节MMPs及其抑制剂来实现的。综上所述,本文首次将脂肪细胞因子衍生物抵抗素13肽用于抗肿瘤侵袭迁移研究,发现抵抗素13肽在体内外抑制肿瘤细胞的增殖、粘附和侵袭迁移能力,并初步认识其作用机制。同时在细胞水平证实另一种脂肪细胞因子衍生物脂联素重组体也能抑制肿瘤细胞的粘附及侵袭迁移能力。这有利于从脂肪细胞因子抗肿瘤侵袭迁移作用的角度,拓展脂肪细胞因子抗肿瘤及治疗其他疾病机制研究的范围,并为开发脂肪细胞因子类药物抑制肿瘤侵袭转移等疾病提供新的理论依据。更多还原

【Abstract】 Obesity is a serious health problem, as it is not only associated with a variety of metabolic disorders but also with an increased risk of developing cancer. More specifically, obesity has been identified as a risk factor for breast cancer. Unfortunately, the mechanism by which obesity increases cancer risk is not yet known. Insights into the mechanism(s) through which obesity increases the risk of cancer are urgently needed to develop new strategies for the prevention and treatment of certain cancers. Current epidemiologic and experimental studies of the obesity cancer association focus on the role of increased adipose tissue, particularly the increase in circulating adipocyte-derived factors (adipokines). Multiple factors from adipose tissue, such as leptin, adiponectin, cytokines, and other secreted products, influence processes involved in carcinogenesis.Metastasis is one of the major causes of mortality in breast cancer patients and occurs as a complex multistep process that involves cancer cell adhesion, invasion, and migration. In the multiple stages of these processes, the degradation of environmental barriers, such as the extracellular matrix (ECM) and basement membrane, is the initial step, and several proteolytic enzymes participate in the degradation of these barriers. Among these enzymes, MMPs play a major role. TIMPs are natural inhibitors of MMPs found in most tissues and body fluids. By inhibiting MMPs activities, they participate in tissue remodeling of the ECM and inhibit tumor invasion and metastasis. Numerous investigations have pointed out that the MMPs and TIMPs play an important role in the invasion and metastasis of cancers. In addition, MMP-2 and MMP-9 degrade components of the basement membrane and are strongly implicated in the invasion and metastasis of malignant tumors. Therefore, the inhibition of invasion mediated by MMP-2 and MMP-9 may be crucial for the inhibition of cancer metastasis. Recent clinical studies have shown that MMP activity is required for rapidly proliferating and invading tumors rather than for already established tumors. Thus, the inhibition of MMP activity is important for the prevention of early stage carcinogenesis, particularly the tumor promotion process.Resistin is known as an adipocyte-specific secretory cysteine-rich hormone that can cause insulin resistance and decrease adipocyte differentiation. Many aspects studies have provided evidence of a role of resistin in inflammatory processes that may be involved in atherosclerosis. The aim of this study was to elucidate the potential of resistin-13-peptide as an inhibitor of MMP-2 and MMP-9 activities using gelatin zymography, and to present the expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 proteins using western blotting. We also investigated the influence of resistin-13-peptide on cancer cell viability, colony formation, adhesion, invasion, and animal experiment using MDA-MB-231 breast cancer cells. In results, Resistin-13-peptide inhibited MDA-MB-231 cells growth and colony formation in dose-dependent and time-dependent manners. Meanwhile, the invasive and adhesive abilities of MDA-MB-231 cells were yet cut down by resistin-13-peptide in dose-dependent manners. Resistin-13-peptide decreased the gelatinolytic activities of both MMP-2 and MMP-9, enhanced the protein expression of TIMP-1 and TIMP-2, which were secreted from the MDA- MB-231 cells. The animal experiments found the growth of tumors were repressed by resistin-13- peptide, which not injured other organs in the same time. Especially ovaries didn’t have pathological changes yet. In conclusions, the treatment with resistin-13- peptide is effective in suppressing tumor proliferation, adhesion and invasion. The possible mechanism is down-regulation of MMPs and up-regulation of TIMPs.In addition, adiponectin, a major adipose cytokine, plays a crucial role in the inhibition of metabolic syndrome by acting on such cell types as muscle cells and hepatocytes. Furthermore, evidence suggests that adiponectin may influence cancer pathogenesis. Previous studies have been reported on the potential link between breast cancer and adiponectin in the epidemiologic surveys. Low serum adiponectin levels may be a novel risk factor for breast cancer and study of adiponectin biology can provide new insights into the association of obesity with cancer risk. Since the mechanisms of action of adiponectin are not entirely clear, future studies are needed to fully elucidate the action of this hormone. In the present study, we investigated the influence of g-adiponectin, which is human adiponectin globular domain (104-247) recombinant protein, on cancer cell viability, adhesion, invasion and motility using MDA-MB-231 cells. Furthermore, we determined the potential effects of g-adiponectin on MMP-2 and MMP-9 activities using gelatin zymography, and to present the expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 proteins and mRNA using western blotting and RT-PCR assays. In results, adiponectin recombinant inhibited MDA- MB-231 cells growth and colony formation in dose-dependent and time-dependent manners. Meanwhile, the invasive and adhesive abilities of MDA-MB-231 cells were yet cut down by adiponectin recombinant in dose-dependent manners. Adiponectin recombinant decreased the gelatinolytic activities of both MMP-2 and MMP-9, enhanced the protein expression of TIMP-1 and TIMP-2, which were secreted from the MDA- MB-231 cells. This was testified in mRNA level in the same time. In conclusions, the treatment with adiponectin recombinant is effective in suppressing tumor proliferation, adhesion and invasion. The possible mechanism is down-regulation of MMPs and up-regulation of TIMPs.更多还原