Co-expression of urokinase-type plasminogen activator and its receptor in human gastric-cancer cell lines correlates with their invasiveness and tumorigenicity

The expression of urokinase-type plasminogen activator (u-PA), its receptor (u-PAR) and metalloproteases activity were analyzed in 4 human gastric-cancer cell lines (AGS, Hs746T, SNU-1, and SNU-5), in an attempt to relate these activities to their invasive potential and tumorigenicity on the modified chorioallantoic membranes (CAM) of chick embryos. Only 1 of the 4 cell lines tested, Hs746T, expressed both u-PA and u-PAR as well as MMP-2, but not MMP-9. This cell line was both tumorigenic and highly invasive (51.3 ± 13.1%) on a modified CAM. Its invasive capacity was comparable with that of a highly malignant human epidermoid-carcinoma cell line (HEp3), which usually showed 40 to 50% invasiveness. The 3 other cell lines all produced MMP-2 and MMP-9, but only AGS showed moderate invasiveness (24.2 ± 8.8%). While antibodies to u-PA were significantly effective in reducing CAM invasiveness of Hs746T cells by approximately 40%, the invasiveness of the t-PA-expressing AGS cell line was not affected by anti-t-PA antibodies. These results suggest that when one of the components of the u-PA/u-PAR system (the enzyme and/or the receptor) is not produced and u-PA/u-PAR-dependent cell-surface proteolytic activity is thereby diminished, the malignant phenotype that can be determined by tumorigenicity and invasion of connective tissue on a CAM is compromised. Production of both type-IV collagenases (MMP-2 and MMP-9) cannot offset this deficiency.Int. J. Cancer 71: 867-873, 1997. © 1997 Wiley-Liss Inc.     

Decrease of breast cancer cell invasiveness by sodium phenylacetate (NaPa) is associated with an increased expression of adhesive molecules

Sodium phenylacetate (NaPa), a non-toxic phenylalanine metabolite, has been shown to induce in vivo and in vitro cytostatic and antiproliferative effects on various cell types. In this work, we analysed the effect of NaPa on the invasiveness of breast cancer cell (MDA-MB-231, MCF-7 and MCF-7 ras). Using the highly invasive breast cancer cell line MDA-MB-231, we demonstrated that an 18-hour incubation with NaPa strongly inhibits the cell invasiveness through Matrigel (86% inhibition at 20 mM of NaPa). As cell invasiveness is greatly influenced by the expression of urokinase (u-PA) and its cell surface receptor (u-PAR) as well as the secretion of matrix metalloproteinases (MMP), we tested the effect of NaPa on these parameters. An 18-hour incubation with NaPa did not modify u-PA expression, either on MDA-MB-231 or on MCF-7 and MCF-7 ras cell lines, and induced a small u-PA decrease after 3 days of treatment of MDA-MB-321 with NaPa. In contrast, an 18 h incubation of MDA-MB-231 increased the expression of u-PAR and the secretion of MMP-9. As u-PAR is a ligand for vitronectin, a composant of the extracellular matrix, these data could explain the increased adhesion of MDA-MB-231 to vitronectin, while cell adhesivity of MCF-7 and MCF-7 ras was unmodified by NaPa treatment. NaPa induced also an increased expression of both Lymphocyte Function-Associated-1 (LFA-1) and Intercellular Adhesion Molecule-1 (ICAM-1), which was obvious from 18 hour incubation with NaPa for the MDA-MB-231 cells, but was delayed (3 days) for MCF-7 and MCF-7 ras. Only neutralizing antibodies against LFA-1 reversed the decreased invasiveness of NaPa-treated cells. Therefore we can conclude that the strong inhibition of MDA-MB-231 invasiveness is not due to a decrease in proteases involved in cell migration (u-PA and MMP) but could be related both to the modification of cell structure and an increased expression of adhesion molecules such as u-PAR and LFA-1.     

Inhibition of urokinase plasminogen activator with a novel enzyme inhibitor, WXC-340, ameliorates endotoxin and surgery-accelerated growth of murine metastases

The urokinase plasminogen activator (u-PA) is intimately associated with tumour invasion and metastases. Surgery facilitates accelerated metastatic tumour growth in murine models, a phenomenon related to elevated perioperative bacterial lipopolysaccaride (LPS) and inflammatory cytokine levels. The objectives of the study were to examine the role of u-PA in cytokine-enhanced tumour cell invasion in vitro and surgery-induced accelerated metastatic tumour growth in vivo and to assess the potential benefit of a novel selective u-PA inhibitor WXC-340 in this setting. CT-26 murine colorectal carcinoma cells were stimulated with LPS, tumour necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6). Cell supernatant u-PA expression and activity were determined using a colorimetric assay and Western blot analysis, respectively. Baseline and cytokine-stimulated in vitro invasion were assessed using ECmatrix invasion chambers. Two established murine models of accelerated metastatic tumour growth were used to investigate the consequences of u-PA inhibition on postoperative metastatic tumour burden. The effect of u-PA inhibition in vitro and in vivo was examined using the novel selective u-PA inhibitor, WXC-340. Proinflammatory cytokine stimulation significantly enhanced in vitro u-PA expression, activity and extracellular matrix invasion by approximately 50% compared to controls (P<0.05). This was abrogated by WXC-340. In vivo WXC-340 almost completely ameliorated both LPS- and surgery-induced, metastatic tumour growth compared to controls (P>0.05). In conclusion, u-PA cascade is actively involved in cytokine-mediated enhanced tumour cell invasion and LPS and surgery-induced metastatic tumour growth. Perioperative u-PA inhibition with WXC-340 may represent a novel therapeutic paradigm.     

Stable expression of antisense urokinase mRNA inhibits the proliferation and invasion of human hepatocellular carcinoma cells

Urokinase-type plasminogen activator (u-PA) plays a key role in malignant tumor behavior. We have previously shown that the expression of high levels of u-PA mRNA in human hepatocellular carcinoma (HCC) biopsies was inversely correlated with the survival of the patients. In order to evaluate the involvement of u-PA in the invasive and infiltrating properties of HCC cells, the SKHep1C3 cell line was stably transfected with an expression vector containing the 5' portion (257 bp) of u-PA cDNA in the antisense orientation. u-PA mRNA expression and its protein level and enzymatic activity were specifically inhibited in the antisense transfectants. A comparable inhibition of the u-PA receptor (u-PAR) mRNA and protein was also evidenced in the antisense transfected cells compared with the control ones. At the functional level, the SKHep1C3-AS cells showed a significant reduction in proliferation, Matrigel invasion, and motility assays compared to parental and vector-alone cells. These results indicate that u-PA is an essential factor in the growth and invasiveness of human hepatocarcinoma cells. Antisense u-PA strategy might be a potential approach to reduce tumor growth as well as the invasive capacity of the malignant cells in HCC.     

Glioma-conditioned medium blocks endothelial cells’ apoptosis Induced by hypoxia and promotes its angiogenesis via up-regulation of u-PA/u-PAR

Objective: To investigate the role of urokinase-type plasminogen activator/urokinase-type plasminogen receptor(u-PA/u-PAR) system in glioma angiogenesis under hypoxic conditions, we studied the effect of glioma-conditioned medium on the hypoxia induced changes in human endothelial-like ECV304 cells proliferation, apoptosis, cord formation in vitro and u-PA/u-PAR expression.Methods: MTT assay was used to examine the changes in cell proliferation. Cell apoptosis was analyzed by Hoechst 33258 staining. Matrigel cord-like formation assay was used to evaluate the angiogenesis ability of ECV304 cells in vitro. Expressions of u-PA/u-PAR mRNA were detected by quantitative real-time RT-PCR.Results: Hypoxia inhibited ECV304 cells proliferation and induced cell apoptosis. Hypoxic conditioned medium(H-CM) while not normoxic conditioned medium(N-CM) of U251 glioma cells partially blocked the effect of hypoxia on ECV304 cells proliferation and apoptosis. H-CM of U251 glioma cells also promoted the cord formation of ECV304 cells seeded on matrigel. When u-PA or u-PAR monoclonal antibodies were added into ECV304 cells culturing medium, cord formation ability was partially inhibited. H-CM of U251 glioma cells induced uPA and uPAR expression in ECV304 cells.Conclusion: These suggest that u-PA/u-PAR system is involved in glioma angiogenesis trigged by hypoxic microenviroment.     

The urokinase-type plasminogen activator, its receptor and u-PA inhibitor type-1 affect in vitro growth and invasion of Kaposi's sarcoma and capillary endothelial cells: role of HIV-Tat protein

The aggressive and malignant nature of AIDS-associated Kaposi's sarcoma (KS) lesions have largely been ascribed to Tat, the HIV-1 transactivator protein. Among other activities, HIV-Tat induces the migration and invasion of KS and endothelial cells. Since cell invasion is strictly correlated to the activity of lytic enzymes, we elucidated the role of the cell-associated plasminogen activation system in Tat-dependent and in constitutive invasion and proliferation of KS and of microvascular endothelial cells (MVEC). We demonstrate that KS cells and MVEC express the u-PA receptor (u-PAR) and release plasminogen activators and plasminogen activator inhibitor type-1 (PAI-1). The urokinase-type plasminogen activator (u-PA) is chemotactic, chemoinvasive and mitogenic for KS cells and for MVEC. Conditioned medium from KS cells induced invasion and proliferation of MVEC through the u-PA/u-PAR system. Tat is motogenic and mitogenic on KS cells and MVEC, and stimulates morphogenesis of MVEC. These activities were inhibited following antagonization of u-PA and u-PAR, which also reduced constitutive proliferation and invasion of KS cells and MVEC. These data indicate that the u-PA/u-PAR/PAI-1 system is involved in KS-induced endothelial cell invasion, proliferation, and differentiation. Further, exogenous Tat protein could up-regulate the fibrinolytic system, increasing its influence on KS and endothelial cell proliferation and migration, potentially promoting KS progression. These observations suggest the potential for application of u-PA/u-PAR system inhibitors for control of AIDS-associated KS, that has a high risk of recurrence with highly active antiretroviral therapy failure, and of other KS forms.     

Fascin-1 expression correlates with repression of E-cadherin expression in hepatocellular carcinoma cells and augments their invasiveness in combination with matrix metalloproteinases

Expression of fascin-1, an actin bundling protein, is a poor prognostic factor in hepatocellular carcinoma (HCC). However, its biological role in HCC cells remains unclear. Using human HCC tissues and cell lines HLE, Hep3B, and Huh7, we investigated whether fascin-1 is involved in epithelial-mesenchymal transition (EMT) and increases invasiveness, thus serving as a promoter of cancer aggressiveness. Immunohistochemical analysis revealed that fascin-1 expression in 19% of primary HCCs was associated with repression of E-cadherin expression, indicating EMT. In vitro, HLE cells showed high fascin-1 expression, loss of E-cadherin, and efficient invasion through Matrigel. Knockdown of fascin-1 significantly repressed invasiveness of the HLE cells and slightly induced E-cadherin expression. In contrast, Huh7 cells had low fascin-1 levels, high E-cadherin expression, and were expectedly non-invasive. However, forced overexpression of fascin-1 conferred only modest invasiveness without E-cadherin repression, indicating that fascin-1 alone cannot effectively stimulate invasiveness or EMT. Furthermore, Hep3B cells were non-invasive despite high fascin-1 expression. Nevertheless, fascin-1 overexpression dramatically increased the migratory potential of Huh7 cells. We then evaluated matrix metalloproteinases (MMPs) 2 and 9 from the HCC cell lines. Significant MMP secretion was only found in HLE cells. Although MMP levels were not elevated in fascin-1-overexpressing Huh7 cells, their invasiveness was remarkably augmented by coculture with HLE cells, and was suppressed in the presence of an MMP inhibitor. In conclusion, we propose that fascin-1 primarily acts as a migration factor associated with EMT in HCC cells and facilitates their invasiveness in combination with MMPs.     

乳腺癌纤溶分子标志物t-PA、u-PA表达的临床研究

目的:检测乳腺癌患者血浆纤溶分子标志物含量、mRNA水平的变化以探索其与肿瘤浸润、播散的关系及临床意义。方法:采用逆转录、实时定量PCR技术检测30例乳腺癌组织中组织型纤溶酶原激活剂(tPA)、尿激酶型纤溶酶原激活剂(uPA)mRNA水平;用ELISA法同步检测患者血浆纤溶分子标志物含量,包括tPA、uPA、尿激酶型纤溶酶原激活剂受体(uPAR)、纤溶酶抗纤溶酶复合物(PAP)等。结果:乳腺癌患者血浆uPA、uPAR、PAP含量均较正常对照明显升高,其中,有周围淋巴结转移、远处脏器转移者uPA升高更为显著。肿瘤细胞uPAmRNA表达增高,在不同临床分期组中有显著差异,而tPAmRNA则减少。雌孕激素受体双阳性组tPAmRNA表达较其他组为高。结论:乳腺癌患者纤溶功能明显亢进,可能是其易播散、浸润的主要原因之一。逆转录实时定量PCR技术,使tPAmRNA、uPAmRNA有望成为临床上乳腺癌病情监测、预后判断及治疗选择的辅助指标。     

Relation of type II transforming growth factor-beta receptor to hepatic fibrosis and hepatocellular carcinoma

Hepatocarcinogenesis is closely related to hepatic fibrosis. In this study, we investigated the relationship of type II transforming growth factor-beta receptor (T beta RII) to hepatic fibrosis and hepatocellular carcinoma (HCC). In vivo: liver tissues were obtained from 30 patients (10 chronic hepatitis, 7 cirrhosis, 13 HCC). Protein expression and immunolocalization of T beta RII were examined by Western blot analysis and immunohistochemistry. In vitro: T beta RII protein expression in hepatoma cell lines (HepG2, Hep3B, HLE, HLF and Huh7) was examined by Western blot analysis. Next, we transfected T beta RII cDNA to Huh7, and compared the change of cell number and observed the induction of apoptosis after TGF-beta1 treatment using a FACScan flow cytometer. In vivo: T beta RII immunolocalization in liver tissues was significantly decreased in patients with HCC compared with that of patients with chronic hepatitis or liver cirrhosis. In Western blot analysis, T beta RII expression in tissues attenuated in comparison with that in non-tumor tissues in some patients with HCC. In vitro: T beta RII protein expression in HLE, HLF and Huh7 cells was weaker than that in HepG2 and Hep3B cells. In Huh7 cells transfected T beta RII cDNA, cell arrest and apoptosis were obviously induced. These results indicated that human HCC has a reduced expression of T beta RII for TGF-beta1. This may provide a selective growth advantage to HCC to escape the inhibitory growth signals of TGF-beta1, and may be linked with critical steps in the growth of hepatoma cells.     

In vivo and in vitro invasion in relation to phenotypic characteristics of human colorectal carcinoma cells.

In this study we investigated the tumorigenicity, growth pattern and spontaneous metastatic ability of a series of nine human colorectal carcinoma cell lines after subcutaneous and intracaecal xenografting in nude mice. CaCo2 cells were found to be poorly tumorigenic to non-tumorigenic in either site; the other cell lines were tumorigenic in both sites. SW1116, SW480 and SW620 did not show local invasive in the NCI-H716 and LS174T cells were both invasive in the caecum, but only NCI-H716 was invasive in the subcutis. HT29 and 5583 (S and E) cells were invasive in the caecum and from that site metastatic to the lungs and/or the liver. HT29 and 5583S cells were both invasive in the subcutis, but 5583E cells were not. Of each category of in vivo behaviour in the caecum, one cell line was further investigated with regard to invasion in vitro (into embryonic chick heart fragments), E-cadherin expression in vivo and in vitro and in vitro production of u-PA and t-PA. These parameters were chosen in view of their purported role in extracellular matrix degradation and intercellular adhesion, which are all involved in the invasive and metastatic cascade. Invasion in vitro was not predictive for invasion or metastasis in vivo. In the cell line which showed invasion in embryonic chick heart tissue, heterogeneous E-cadherin expression was observed in vitro together with a relatively high production of u-PA. The non-invasive cell lines showed in vitro homogeneous expression of E-cadherin with a relatively low production of u-PA. In vivo expression of E-cadherin was either absent or heterogeneous. We conclude that: (1) colorectal carcinoma xenografts show site-specific modification of in vivo invasive and metastatic behaviour; (2) invasion in vitro does not correlate with invasion and metastasis in vivo; (3) in vitro non-invasion might be associated with homogeneous E-cadherin expression and low production of u-PA; (4) E-cadherin expression in vitro differs from E-cadherin expression in vivo. The results support the notion that the microenvironment in which cancer cells grow is one of the factors involved in the regulation of invasive and metastatic behaviour.     

Endogenous receptor-bound urokinase mediates tissue invasion of the human lung carcinoma cell lines A549 and Calu-1.

Macrophage colony-stimulating factor (CSF-1) increases the tissue invasive potential of the CSF-1 receptor-bearing lung carcinoma cell lines A549 and Calu-1 by increasing the number of endogenously bound urokinase-type plasminogen activators (u-PA)s on these cells. CSF-1, at concentrations which optimize invasion of A549 and Calu-1 cells into human amnion membranes (250 ng/ml), maximally augments the number of u-PA receptors occupied by endogenously produced urokinase. Preincubation of A549 and Calu-1 cells with the anti-u-PA monoclonal antibody MPW5UK (25 micrograms/ml) or with a 20- to 40-fold stoichiometric excess of fluid phase type 2 plasminogen activator inhibitor abrogates invasiveness, indicating that functionally active cell surface u-PA is essential for tissue invasion. In contrast, fluid phase type 1 plasminogen activator inhibitor (PAI-1, 5-15 units/ml) does not inhibit invasiveness unless preincubated with the amnion membranes. Inhibition of invasion by PAI-1 is abolished by presaturating the amnion membranes with antiitronectin monoclonal antibody (10 micrograms/ml) which prevents binding of PAI-1 to tissue-associated vitronectin. Binding of PAI-1 to tissue vitronectin is therefore a prerequisite for its inhibitory action. Thus, endogenously receptor-bound u-PA is the primary protease mediating CSF-1-induced tissue invasiveness of the lung carcinoma cell lines A549 and Calu-1.     

A peroxisome proliferator-activated receptor gamma antagonist induces vimentin cleavage and inhibits invasion in high-grade hepatocellular carcinoma

Increased expression of vimentin in carcinomas correlates with parameters of malignant potential such as tumor grade and tumor metastasis. Peroxisome proliferator-activated receptor gamma (PPARgamma) has been intensively evaluated as a potential target for the inhibition of cell growth and metastasis in cancer cells. In the present study, we examined whether PPARgamma is a possible target molecule for the prevention of cell growth and invasion by treatment with agonists (troglitazone, rosiglitazone) and antagonists (T0070907, GW9662) in four different hepatocellular carcinoma (HCC) cell lines. We also evaluated the effects of the PPARgamma agonists and antagonists on tumor cell migration and invasion. The expression level of PPARgamma protein was higher in the sarcomatoid SH-J1 and poorly differentiated HLE cell lines than that in the well-differentiated HCC cell lines (HepG2 and Huh-7). Expression of vimentin was high in the SH-J1 HCC cell line and minimally detected in the HLE cell line. Treatment with low doses of the PPARgamma antagonists inhibited cell growth and colony formation of all four of the HCC cell lines. Vimentin in the high-grade HCC cells was cleaved by the treatment with the PPARgamma antagonists. Furthermore, treatment with the PPARgamma antagonists also strongly inhibited migration and invasion of the SH-J1 and HLE cells. However, treatment with low doses of the agonists had no effect on vimentin expression, migration, and invasion of the high-grade HCC cells but cell growth was inhibited by treatment with high concentrations of the agonists. Our results indicate that treatment with a PPARgamma antagonist may prevent cell growth and invasion of high-grade HCC cells. Our findings also suggest that PPARgamma antagonists inhibit cell growth and invasion through vimentin disarrangement in high-grade HCC.     

The soluble form of human leukocyte antigen class I antigen causes apoptosis on hepatocellular carcinoma cell lines

A soluble form of human leukocyte antigen class I antigen (sHLA-I) has been reported to cause apoptosis on cytotoxic T cells and inhibit killer activity of natural killer cells via killer-cell inhibitory receptors. However, its effect on cancer cells has not yet been elucidated. We examined the direct effect of sHLA-I on human liver cancer cell lines, HepG2, HLE and HLF. The effects of sHLA-I on cell growth, DNA synthesis, and apoptosis induction were evaluated. To elucidate the mechanisms, cDNA expression arrays were also examined. sHLA-I caused cell growth inhibition, resulting in apoptosis on human hepatocellular carcinoma, dose-dependently. In this process, caspase-3 was activated. sHLA-I also inhibited in vivo growth of hepatocellular carcinoma in severe combined immunodeficient mice. sHLA-I caused apoptosis on human hepatocellular carcinoma.     

泰素帝对胰腺癌细胞侵袭能力的影响

目的 研究泰素帝（TXT）在体外对肿瘤细胞的运动，黏附及侵袭能力的影响。探讨TXT治疗胰腺癌的作用。方法 用Transwell小室重建基底膜侵袭模型。趋化运动模型及采用细胞黏附分子方法检测TXT对胰腺癌细胞株SUIT-2及其药细胞株S2/TXT的侵袭，运动及黏附能力的影响。结果 TXT可明显抑制SUIT-2细胞的侵袭及运动能力，但对其黏附能力无明显影响。与SUIT-2细胞相比，TXT对S2/TXT细胞的侵袭，运动及黏附能力均无明显作用。结论 TXT可降低胰腺癌细胞的体外侵袭能力。但对胰腺癌耐药细胞株的侵袭能力无明显作用。     

原位杂交检测骨巨细胞瘤u-PA、u-PAR和PAI-2 mRNA的表达及意义

目的 研究骨巨细胞瘤（giant cell tumors of bone,GCT)组织u-PA、u-PAR和PAI－2mRNA的表达与GCT病理分级和复发的关系。方法 用原位杂交技术检测42例GCT组织u-PA和u-PAR和PAI－2 mRNA的表达。结果 （1）u-PA、u-PAR和PAI－2mRNA在GCT组织多核巨细胞（MGC）的阳性率分别为64．3％、71．4％和40．5％;单核基质细胞（MC）的阳性率分别为54．8％、45．2％和9．5％。（2）Ⅱ、Ⅲ级和复发组GCT的MGC u-PA mRNA与MC u-PA、u-PAR mRNA的表达率均明显高于Ⅰ级和无复发组（P均＜0．05）。（3）GCT组织两种细胞间的u-PA mRNA表达、MGC u-PAR与PAI－2 mRNA的表达间、MCu-PA与u-PAR mRNA的表达间均呈正相关;MGC PAI－2mRNA的阳性率明显高于MC的阳性率（P均＜0．05）。结论 u-PA、u-PAR mRNA在GCT组织中的表达,尤其是在MC中的表达,提示肿瘤分化较低和有复发倾向;MC可能是GCT中主要的肿瘤细胞。