非霍奇金淋巴瘤外周血尿激酶型纤溶酶原激活物及其受体测定的临床意义

 目的 研究非霍奇金淋巴瘤（NHL）患者血中尿激酶型纤溶酶原激活物（uPA）、尿激酶型纤溶酶原激活物受体（uPAR）、纤溶酶原激活剂抑制物-1（PAI-1）水平与NHL发生、发展及预后的关系。方法 采用ELISA方法检测38例初治NHL患者血中uPA,uPAR 及PAI-1水平,分析与不同临床分期、治疗效果、乳酸脱氢酶（LDH）的相关性。结果 NHL患者血uPA,uPAR及PAI-1水平均高于正常对照组（P＜0.05）, NHL患者LDH值增高组与LDH正常组比较uPAR增高（P＜0.05）,uPA,uPAR与LDH三者之间呈正相关（P＜0.01）。结论 uPA及可溶性uPA 受体（suPAR）在NHL的发生、发展中起作用,suPAR可以作为判断NHL肿瘤负荷和预后的指标。     

Retinoic acid-enhanced invasion through reconstituted basement membrane by human SK-N-SH neuroblastoma cells involves membrane-associated tissue-type plasminogen activator

Al-trans retinoic acid (RA) enhanced human, S-type, SK-N-SH neuroblastoma cell invasion of reconstituted basement membrane in vitro but did not induce terminal differentiation of this cell line. In contrast to basal invasion, which was urokinase (uPA)- and plasmin-dependent, RA-enhanced invasion was dependent on tissue-type plasminogen activator (t-PA) and plasmin activity. Neither basal nor RA-enhanced invasion involved TIMP-2 inhibitable metalloproteinases. Enhanced invasion was associated with the induction of t-PA expression, increased expression of the putative t-PA receptor amphoterin, increased association of t-PA with cell membranes and increased net membrane-associated PA activity. Enhanced invasion was not associated with significant changes in the expression of uPA or its membrane receptor UPAR; plasminogen activator inhibitors PAI-1 and PAI-2; metalloproteinases MMP-1, MMP-2, MMP-3, MMP-9 and membrane type MMP1; or tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2. RA stimulated the association of t-PA with the external cell membrane surface, which could be inhibited by heparin sulphate but not by mannose sugars or chelators of divalent cations, consistent with a role for amphoterin. Our data indicate that RA can promote the malignant behavior of S-type neuroblastoma cells refractory to RA-mediated terminal differentiation by enhancing their basement membrane invasive capacity. We suggest that this results from the action of a novel, RA-regulated mechanism involving stimulation of t-PA expression and its association with the cell membrane leading to increased PA-dependent matrix degradation. Int. J. Cancer 73:740–748, 1997. © 1997 Wiley-Liss, Inc.     

The relationship of plasminogen activators and oncogenes to tumour invasion

Accruing evidence suggests an association between increased activity of plasminogen activators and transformed cells, and urokinase activity with tumour aggressiveness. The PIP2 pathway seems to provide a link between oncogene-associated transformation, cellular proliferation, plasminogen-activator expression and tumour invasion and metastasis.     

Urokinase-type plasminogen activator and its inhibitor plasminogen activator inhibitor-1

Tumor associated proteolysis is an essential mechanism in invasion and metastasis of cancer. The influence of the serine protease urokinase-like plasminogen activator (u-PA) and its inhibitor plasminogen activator inhibitor-1 (PAI-1) on the clinical prognosis of squamous carcinoma of the head and neck region (HNSCC) was evaluated. U-PA and PAI-1 levels were measured in tumor biopsies of 41 HNSCC patients and 6 biopsies of healthy oral mucosa using ELISA technique. Patients were followed for an average of 24 months. U-PA concentration in tumor tissue was four times higher than in healthy mucosa (4.96 ng/mg protein versus 1.32 ng/mg). PAI-1 levels were 22 times higher (69.55 ng/mg versus 3.18 ng/mg). Univariate Cox regression analysis revealed significant correlation (p=0.022) of PAI-1 with recurrence of the disease and no significance for u-PA. PAI-1 might become a new functional risk factor reflecting clinical prognosis.     

Urokinase plasminogen activator receptor induced non-small cell lung cancer invasion and metastasis requires NHE1 transporter expression and transport activity

Background

Non-small cell lung cancers (NSLC) are aggressive cancers that are insensitive to chemotherapies and accounts for nearly 33% of all cancer deaths in the United States. Two hallmarks of cancer that allow cells to invade and metastasize are sustained proliferation and enhanced motility. In this study we investigate the relationship between urokinase plasminogen activator (uPA)/uPA receptor (uPAR) signaling and Na⁺/H⁺ exchanger isoform 1 (NHE1) expression and activity.

Methods and results

The addition of 10nM uPA increased the carcinogenic potential of three NSCLC cell lines, NCI-H358, NCI-H460, and NCI-H1299. This included an increase in the rate of cell proliferation 1.6 to 1.9 fold; an increase in the percentage of cells displaying stress fibers 3.05 to 3.17 fold; and an increase in anchorage-independent growth from 1.64 to 2.0 fold. In each of these cases the increase was blocked when the experiments were performed with NHE1 inhibited by 10 ??M EIPA (ethylisopropyl amiloride). To further evaluate the role of uPA/uPAR and NHE1 in tumor progression we assessed signaling events using full-length uPA compared to the uPA amino terminal fragment (ATF). Comparing uPA and ATF signaling in H460 cells, we found that both uPA and ATF increased stress fiber formation approximately 2 fold, while uPA increased matrix metalloproteinase 9 (MMP9) activity 5.44 fold compared to 2.81 fold for ATF. To expand this signaling study, two new cell lines were generated, one with reduced NHE1 expression (H460 NHE1 K/D) and one with reduced uPAR expression (H460 uPAR K/D). Using the K/D cell lines we found that neither uPA nor ATF could stimulate stress fiber formation or MMP9 activity in cells with dramatically decreased NHE1 or uPAR expression. Finally, using in vivo tumor formation studies in athymic mice we found that when mice were injected with H460 cells 80% of mice formed tumors with an average volume of 390 mm³. This was compared to 20% of H460 uPAR K/D injected mice forming tumors with an average volume of 15 mm³ and 10% of H460 NHE1 K/D injected mice forming tumors with an average volume of 5 mm³.

Conclusion

Taken together, these data demonstrate that uPA/uPAR-mediated tumor progression and metastasis requires NHE1 in NSCLC cells and suggests a potential therapeutic approach to blocking cancer progression.     

Inhibition of invasion and metastasis in oral cancer by targeting urokinase-type plasminogen activator receptor

There have been reports of strong correlations between poor prognosis in various cancers and concomitant expression of urokinase-type plasminogen activator (uPA) and its surface receptor (uPAR). We and others have previously shown that the uPA system plays a significant role in a subset of head and neck squamous cell carcinoma. In the present study, we found that uPAR is required for invasion and metastasis of highly malignant oral cancer cells (OSC-19). Treating OSC-19 cells with antisense oligonucleotides (AS) targeting uPAR resulted in a dramatic decrease of uPAR mRNA expression. Furthermore, pretreatment with AS or siRNA targeting uPAR inhibited progression of OSC-19 cells in experimental models. These results suggest that overexpression of uPAR increases the invasiveness and metastasis of OSC-19 cells, and that uPAR is a promising therapeutic target for regulation of progression of oral cancer.     

In vitro plasminogen activator activity in human brain tumors

Cell cultures were prepared from nine human brain tumors. Fibrin plate assays showed plasminogen-dependent fibrinolytic activity in lysates and in material released by these neoplastic cells but not in those from normal adult human white matter. Antibodies against human urokinase caused catalytic inhibition of the urokinase and of the plasminogen activator from WI-38 cells, simian virus 40-transformed WI-38 cells, human prostatic cells, and human ovarian carcinoma cells. However, the anti-urokinase immunoglobulin G did not inhibit the plasminogen activator activity of any of the human brain tumor preparations. These studies indicate that the plasminogen activator produced by human brain tumor cells is antigenically different from the plasminogen activator of other human normal and neoplastic cells.     

Macrophages associated with murine tumours express plasminogen activator activity

The fibrinolytic activity of cancer cells has been repeatedly implicated in mechanisms of local spread and tumour invasiveness. Mononuclear phagocytes associated with solid tumours might also contribute to fibrin dissolution at the tumour/host interface through the expression of plasminogen activator (PA) activity. We have investigated the PA activity of tumour-associated macrophages (TAM) from 4 transplanted murine tumours in syngeneic hosts; peritoneal macrophages (native and thioglycolate-elicited) from both tumour-bearing and control animals were studied as reference cells. TAM from 3 tumours (MSV, mFS6, MN/MCAI) had basal levels of PA activity (20% plasminogen-independent) comparable to or higher than those of thioglycolate-elicited peritoneal macrophages from the same tumour-bearing animals. TAM isolated from 1 tumour (MS2) had a PA which was very low (60% plasminogen-independent), but higher than the activity of unstimulated peritoneal macrophages. Molecular analysis of PA by SDS-PAGE electrophoresis and fibrin autography revealed in all macrophages a single species having an apparent MW of 48 kDA. It thus appears that, in some experimental neoplasms, tumour cell vicinity may represent an in vivo stimulus for macrophage PA expression.     

Angiostatic activity of synthetic inhibitors of urokinase type plasminogen activator

We hypothesize that tumor angiogenesis can be limited by the reduction of enzymatic activity of the urokinase type plasminogen activator. The proposed mechanism is elimination of proteolytic activity by the advancing tip of capillaries which utilize proteolysis to produce space needed for vessel expansion. To test our hypothesis, we have investigated the angiostatic activity of synthetic low molecular weight inhibitors of urokinase: amiloride, benzamidine, EGCG, B428, and B623 using the chicken embryo corioallantoic membrane (CAM) model. We found that all tested inhibitors of urokinase cause a significant reduction of angiogenesis.     

Tumour cell thrombospondin-1 regulates tumour cell adhesion and invasion through the urokinase plasminogen activator receptor

We have previously shown that platelet-produced thrombospondin-1 up-regulates the urokinase plasminogen activator and its receptor and promotes tumour cell invasion. Although tumour cells produce thrombospondin-1 in vivo, they produce only minimal amounts of thrombospondin-1 in vitro. To determine the effect of tumour cell-produced thrombospondin-1 in the regulation of the plasminogen/plasmin system and tumour cell invasion, we studied THBS-1-transfected MDA-MB-435 breast cancer cells that overexpress thrombospondin-1. The role of urokinase plasminogen receptor in thrombospondin-1-mediated adhesion and invasion was studied by antisense inhibition, enzymatic cleavage and antibody neutralization. Tumour cell adhesion to collagen and laminin was evaluated. Tumour cell invasion was studied in a modified Boyden chamber collagen invasion assay. Tumour cell thrombospondin-1 induced a 2-7 fold increase in urokinase plasminogen activator receptor and cell-associated urokinase plasminogen activator expression and a 50-65% increase in cell-associated urokinase plasminogen activator and plasmin activities. Furthermore, tumour cell thrombospondin-1 promoted tumour cell invasion and decreased tumour cell adhesion through up-regulation of urokinase plasminogen activator receptor-controlled urokinase plasminogen activator and plasmin activities. We conclude that tumour cell-produced thrombospondin-1 may play a critical role in the regulation of tumour cell adhesion and tumour cell invasion.     

TF和uPAR在两种乳腺癌细胞系中的表达与细胞侵袭力关系的研究

目的:探讨两种不同乳腺癌细胞系MCF-7和MDA-MB231中组织因子(tissuefactor,TF)和尿激酶型纤溶酶原激活剂受体(urokinase plasminogen activator receptor,uPAR)表达的差异,揭示两者与肿瘤侵袭力关系。方法:用RT-PCR法检测两种乳腺癌细胞系中TF和uPAR mRNA,用流式细胞术(flowcytometry,FCM)检测细胞表面TF和uPAR抗原表达,用改良Bodyen小室法研究细胞侵袭力,t检验差异。结果:MDA-MB231细胞TF、uPAR mRNA和抗原表达明显高于MCF-7细胞,而且MDA-MB231乳腺癌细胞的侵袭力高于MCF-7,转移数目分别为351·67±3·78和154·66±4·72,t=56·35,P=0·000。结论:TF和uPAR表达与乳腺癌细胞侵袭转移能力成正比,可作为肿瘤治疗的靶点。肿瘤防治杂志,2005,12(19):1462-1464     

Association of plasminogen activator activity and steroid receptors in human breast cancer

Plasminogen activating activity (PAA) has been determined in 105 human breast cancer cytosols with a colorimetric assay. PAA has been related to steroid receptors in the same cytosols. A strong positive correlation between PAA and progesterone receptor levels was found (Spearmans coefficient of rank correlation, R = 0.74, P < 0.001). PAA discriminates between progestrone receptor-positive and -negative tumours, irrespective of oestradiol receptor content. It is suggested that PAA is a marker of functional oestradiol receptor and that fibrinolytic activity may be a causative factor in the low metastatic potential of progesterone receptor-positive tumours.     

Maximum effect of urokinase plasminogen activator inhibitors in the control of invasion and metastasis of rat mammary cancer

Experimentally induced pulmonary metastases of mammary cancer in the Fisher 344 rat can be suppressed by the inhibition of urokinase plasminogen activator (uPA). The inhibition of uPA with amiloride or B428 has been shown to be dose dependent. Increased dosage levels of inhibitors might be expected to enhance levels of suppression of metastases. The use of each of these inhibitors at equipotent concentrations that exceeded the doses administered in previous studies failed to eliminate pulmonary metastases. These results demonstrate that a maximum limit is attained for the inhibitory capacities on cells during in vitro invasion or in vivo metastasis. At increased levels, uPA inhibitors continue to suppress, but do not eradicate, experimental pulmonary metastases of MATB cell rat mammary cancer.     

Tissue-type plasminogen activator in breast cancer: relationship with estradiol and progesterone receptors

Plasminogen activator (PA) is an estradiol-inducible enzyme and therefore a potential marker for a functional estradiol receptor (ER) in human breast carcinomas. In this investigation tissue-type PA (t-PA) correlated significantly with both ER and progesterone receptors (PR) in human breast carcinomas. In contrast, neither total PA activity nor urokinase-like PA showed any significant correlation with either ER or PR. Other proteases such as a trypsin-like protease, a chymotrypsin-like protease, and cathepsin B also showed no correlation with ER and PR. It was concluded that the t-PA form of PA may be a marker for a functional ER in breast carcinoma and thus be of value in predicting hormone-dependent breast cancers.