Albo D, Berger D H, Rothman V L, Tuszynski G P
Department of Surgery, Medical College of Pennsylvania and Hahnemann University, Philadelphia, Pennsylvania, 19103, USA.
J Surg Res. 1999 Apr;82(2):331-8. doi: 10.1006/jsre.1998.5578.
We previously showed that thrombospondin 1 (TSP-1) upregulates the plasminogen/plasmin system and promotes breast tumor cell invasion. Preliminary data from our laboratory using neutralizing antibodies suggested that the upregulation in breast tumor cell invasion seen in response to TSP-1 involved the urokinase plasminogen activator receptor (uPAR). To confirm these findings in MDA-MB-231 breast cancer cells, we developed three other strategies to study the role of uPAR in tumor cell adhesion and TSP-1-mediated tumor cell invasion: (a) enzymatic cleavage of uPAR with glycosylphosphatidylinositol-specific phospholipase C; (b) inhibition at the mRNA level with a uPAR antisense construct (cells named LKAS-MDA); (c) inhibition of plasminogen binding with the lysine analogue epsilon-aminocaproic acid. Adhesion to laminin and type I and type IV collagen with and without the addition of epsilon-aminocaproic acid was studied. Tumor cell invasion was studied in a modified Boyden chamber collagen invasion assay. Antisense uPAR inhibition decreased uPAR expression by 48-66% and cell-associated urokinase plasminogen activator (uPA) by 30-68%. Additionally, antisense uPAR inhibition induced a 68-70% reduction in uPA and plasmin activities. Antisense uPAR transfection increased tumor cell adhesion by 46-53%. A similar effect was observed in epsilon-aminocaproic acid-treated MDA-MB-231 cells. TSP-1-mediated tumor cell invasion was almost completely inhibited by either antisense uPAR inhibition or treatment with phospholipase C or epsilon-aminocaproic acid. We conclude that uPAR plays a crucial role in the regulation of tumor cell adhesion and TSP-1-mediated tumor cell invasion.
我们之前的研究表明，血小板反应蛋白1（TSP-1）上调纤溶酶原/纤溶酶系统并促进乳腺肿瘤细胞侵袭。我们实验室使用中和抗体获得的初步数据表明，TSP-1诱导的乳腺肿瘤细胞侵袭上调涉及尿激酶型纤溶酶原激活物受体（uPAR）。为了在MDA-MB-231乳腺癌细胞中证实这些发现，我们开发了另外三种策略来研究uPAR在肿瘤细胞黏附和TSP-1介导的肿瘤细胞侵袭中的作用：（a）用糖基磷脂酰肌醇特异性磷脂酶C对uPAR进行酶切；（b）用uPAR反义构建体在mRNA水平进行抑制（命名为LKAS-MDA的细胞）；（c）用赖氨酸类似物ε-氨基己酸抑制纤溶酶原结合。研究了在添加和不添加ε-氨基己酸的情况下，细胞与层粘连蛋白、I型和IV型胶原的黏附情况。在改良的博伊登室胶原侵袭试验中研究了肿瘤细胞侵袭情况。反义uPAR抑制使uPAR表达降低48 - 66%，细胞相关尿激酶型纤溶酶原激活物（uPA）降低了30 - 68%。此外反义uPAR抑制使uPA和纤溶酶活性降低了68 - 70%。反义uPAR转染使肿瘤细胞黏附增加了46 - 53%。在经ε-氨基己酸处理的MDA-MB-231细胞中也观察到了类似的效果。反义uPAR抑制、磷脂酶C处理或ε-氨基己酸处理几乎完全抑制了TSP-1介导的肿瘤细胞侵袭。我们得出结论，uPAR在肿瘤细胞黏附调节和TSP-1介导的肿瘤细胞侵袭中起关键作用。
