In vitro suppression of urokinase plasminogen activator in breast cancer cells--a comparison of two antisense strategies

High level expression of urokinase plasminogen activator (uPA) has been well documented in a variety of tumors. In breast cancer, expression of uPA is essential for tumor cell invasion, metastasis and proliferation. By contrast, the primary objective of tumor therapy is to reduce the uPA expression level within the tumor, which results in abrogation of proliferation, invasion and metastasizing of the tumor cells. We investigated the effects of uPA on the MDA-MB-231 cell line. MDA-MB-231 cells are highly invasive and express high levels of uPA. In our study, uPA inhibition was achieved by two methodologies: a) stable transfection with an antisense uPA vector, b) transfection with siRNA molecules (small interfering RNA). A plasmid vector was constructed by cloning a uPA-specific cDNA (612 bp) fragment into pBK-CMV plasmid in antisense orientation. In contrast, a double-stranded 21-mer siRNA was designed for targeting uPA. The antisense-transfected cells revealed decreased uPA mRNA and protein as detected by real-time PCR, immunocytochemistry, ELISA, and Western blotting. Moreover, the transfected cells exhibited a significantly reduced proliferation activity as determined by a fluorometric proliferation assay. As a conclusion of our study siRNA-technique is the superior method also regarding time saving for clone selection and instant availability of the transfected cells. Moreover, even if both strategies lead to uPA suppression, a stronger inhibitory effect could be obtained by application of the siRNA-based technique.     

Genetic downregulation of pregnancy-associated plasma protein-A (PAPP-A) by bikunin reduces IGF-I-dependent Akt and ERK1/2 activation and subsequently reduces ovarian cancer cell growth, invasion and metastasis

A Kunitz-type protease inhibitor, bikunin, downregulates expression of uPA and its receptor uPAR at the mRNA and protein levels in several types of tumor cells. Our recent work showed that, using a cDNA microarray analysis, pregnancy-associated plasma protein-A (PAPP-A) is a candidate bikunin target gene. To clarify how reduced levels of PAPP-A may confer repressed invasiveness, we transfected human ovarian cancer cell line HRA with antisense (AS)-PAPP-A cDNA and compared the properties of the transfected cells to those of parental HRA cells. Here, we show that regulation of uPA mRNA and protein by IGF-I depends on the PI3K and MAPK signaling pathways and phosphorylation of Akt and ERK1/2 is required for IGF-I-mediated cell invasion; that IGFBP-4 protease in HRA cells is identified as PAPP-A; that reduced PAPP-A expression is associated with the upregulation of IGFBP-4 expression; that higher intact IGFBP-4 levels were associated with low invasive potential and growth rate in AS-PAPP-A cells in response to IGF-I; that IGF-I stimulates Akt and ERK1/2 activation of both the control and antisense cells, but the relative potency and efficacy of IGF-I were lower in the antisense cells compared to the control; and that genetic downregulation of PAPP-A reduces the proliferation, invasion and metastasis of HRA cells. In conclusion, our data identify a novel role for PAPP-A as a bikunin target gene. IGF-I-induced IGFBP-4 proteolysis by PAPP-A may enhance cell growth and invasion through IGF-I-dependent Akt and ERK1/2 activation and subsequently upregulation of uPA.     

Regulation of the urokinase-type plasminogen activator system by the von Hippel-Lindau tumor suppressor gene.

The urokinase-type plasminogen activator (uPA) system plays an important role in tumor cell invasion, metastases, and angiogenesis. uPA, uPA receptor, and plasminogen activator inhibitor 1 (PAI-1) are prognostic factors in different solid tumors, e.g., renal cell carcinomas (RCCs). von Hippel-Lindau (VHL) disease is an inherited cancer syndrome that is characterized by extensively vascularized tumors, including hemangioblastomas and RCCs. In 75% of sporadic RCCs, the VHL gene is also inactivated. It has been recognized in sporadic RCC that PAI-1 mRNA levels are up-regulated and uPA mRNA levels are down-regulated. We determined the role of the VHL tumor suppressor gene in the regulation of the uPA system in RCC. In 786-O RCC cells expressing the wild-type (wt) VHL gene, we measured a 3-fold higher overall urokinase activity than in 786-O cells expressing a mutant VHL gene or lacking VHL. uPA mRNA and protein levels were higher in cells with wt VHL compared with cells with mutant VHL or lacking VHL. In addition, PAI-1 mRNA and protein levels were dramatically increased in 786-O cells with mutant VHL or lacking VHL, compared with cells expressing wt VHL. Our results provide further evidence that the VHL gene plays an important role in the process of angiogenesis by regulation of plasmin-mediated proteolysis of the extracellular matrix and may explain why VHL-induced RCCs grow slowly and metastasize relatively late.     

Urokinase induces receptor mediated brain tumor cell migration and invasion

The plasminogen activation (PA) system plays an important role in tumor invasion by initiating pericellular proteolysis of the extracellular matrix (ECM) and inducing cell migration. Malignant brain tumors overexpress PA members and characteristically invade by migrating on ECM-producing white matter tracts and blood vessel walls. To determine whether urokinase-type plasminogen activator (uPA) and its receptor (uPAR) directly modulate the migration of brain tumor cells, we examined six human brain tumor cell lines, 2 astrocytomas (SW1088, SW1783), 2 medullobastomas (Daoy, D341Med), and 2 glioblastomas (U87MG, U118MG), for their surface uPAR expression, endogenous PA activity, and functional proteolytic activity by an ECM-degradation assay. Migration on Transwell membranes and invasion of Matrigel was then tested by pre-incubating the cells with increasing concentrations of either uPA, the proteolytically inactive amino-terminal fragment (ATF) of uPA, or the uPAR cleaving enzyme, phosphatidylinositol-specific phospholipase C (PI-PLC).All of the cell lines, except D341Med, express surface uPAR protein and uPA activity. High levels of uPAR and uPA activity correlated with cellular degradation of ECM, cell migration, and Matrigel invasion. Cell migration and invasion were enhanced by uPA or ATF in a dose dependent manner, while PI-PLC treatment abolished the uPA effect and inhibited migration and invasion. We conclude that ligation of uPAR by uPA directly induces brain tumor cell migration, independent of uPA-mediated proteolysis; and in concert with ECM degradation, markedly enhances invasion. Conversely, removing membrane bound uPAR from the surface of the cells studied inhibited their ability to migrate and invade even in the presence of proteolytically active uPA.     

二氟甲基鸟氨酸及反义bcl-2协同诱导HL60细胞凋亡

目的　观察二氟甲基鸟氨酸 (DFMO)及反义bcl 2调控bcl 2基因表达对HL6 0细胞凋亡的诱导作用。方法　构建反义bcl 2逆转录病毒重组体 ,建立产病毒细胞系 ,转染HL6 0细胞 ,用形态观察、生长曲线、FCM分析、集落形成、DNA电泳图谱、分子杂交及免疫组化等方法研究细胞生长特性及细胞凋亡诱导。结果　成功地构建了反义bcl 2逆转录病毒重组体及产病毒细胞系。以此转染HL6 0细胞 ,引起了bcl 2mRNA及蛋白表达下降 ,但生长速率、细胞周期分布及鸟氨酸脱羧酶 (ODC)基因表达水平与亲本细胞比较差别不大 ;无明显的细胞凋亡 ,仅集落形成能力有所减弱。用小剂量DFMO处理反义bcl 2转染的细胞 ,不仅生长受到明显抑制 ,而且可诱导细胞凋亡。结论　DFMO与反义bcl 2对HL6 0细胞增殖抑制及凋亡诱导有协同作用。抑制bcl 2表达能提高肿瘤细胞对DFMO作用的敏感性。     

Synergistic down-regulation of urokinase plasminogen activator receptor and matrix metalloproteinase-9 in SNB19 glioblastoma cells efficiently inhibits glioma cell invasion,angiogenesis, and tumor growth

The binding of urokinase plasminogen activator (uPA) to its receptor (uPAR) initiates a proteolytic cascade facilitating the activation of matrix metalloproteinase-9 (MMP-9), which in turn degrades the extracellular matrix. These processes have an established role in tumor invasion and metastasis. Our previous work revealed an inverse association between glioma invasion and the expression of uPAR and MMP-9. In the present study, we used the adenovirus serotype 5 vector system to generate a replication-deficient recombinant adenovirus capable of simultaneously expressing antisense uPAR and antisense MMP-9 (Ad-uPAR-MMP-9). This adenoviral construct is driven by the independent promoter elements cytomegalovirus and bovine growth hormone and SV40 polyadenylation signals to down-regulate key steps in the proteolytic cascade. Ad-uPAR-MMP-9 infection of SNB19 cells significantly decreased uPAR and MMP-9 expression as determined by immunohistochemical and Western blotting analyses. A Matrigel invasion assay revealed marked reduction in the invasiveness of the Ad-uPAR-MMP-9-infected cells compared with parental and vector controls. Tumor spheroids infected with Ad-uPAR-MMP-9 and cocultured with fetal rat brain aggregates did not invade rat brain aggregates, whereas 90-95% of the mock and empty vector-infected cells invaded the rat brain aggregates. Intracranial injection of SNB19 cells infected ex vivo with the Ad-uPAR-MMP-9 antisense bicistronic construct showed decreased invasiveness and tumorigenicity. s.c. injections of the bicistronic antisense construct into established tumors (U87 MG) caused tumor regression. These results support the therapeutic potential of targeting the individual components of the uPAR-MMP-9 by using a single adenovirus construct for the treatment of gliomas and other cancers.     

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.     

法舒地尔对胃癌MGC-803细胞增殖、侵袭和迁移的影响

目的 探讨法舒地尔对胃癌MGC-803细胞增殖、侵袭和迁移的影响及可能机制。方法 采用不同浓度的法舒地尔处理胃癌MGC-803细胞,检测细胞数目;选择无显著细胞毒性的3组浓度（10、25、50 μmol／L）法舒地尔进行后续实验,将经药物处理的胃癌MGC-803细胞作为处理组,而未经药物处理的胃癌MGC-803 细胞作为对照组;细胞培养0、24、48、72、96 h后,CCK-8法检测细胞增殖倍数;划痕实验检测细胞迁移能力;Transwell法检测细胞侵袭能力;Western blotting检测细胞中血管内皮细胞生长因子（VEGF）、基质金属蛋白酶9（MMP-9）、基质金属蛋白酶14（MMP-14）和上皮间质转化（EMT）的标志蛋白波形蛋白（Vimentin）和上皮型钙黏蛋白（E-cadherin）的表达水平。结果 CCK-8法检测结果显示,法舒地尔以剂量依赖性的方式抑制胃癌MGC-803细胞的生长,法舒地尔浓度越高,处理组的细胞数量越少,增殖倍数明显降低（P＜0.01）;划痕实验结果表明,处理组伤痕愈合率明显较对照组降低,且法舒地尔浓度越高,处理组伤痕愈合率越低（P＜0.01）;Transwell结果显示,与对照组比较,处理组侵袭的细胞数目明显减少（P＜0.01）;与对照组比较,法舒地尔明显下调VEGF、MMP-9、MMP-14和Vimentin蛋白的表达水平,而上调E-cadherin蛋白的表达水平。结论 法舒地尔可抑制胃癌MGC-803细胞的的增殖、迁移和侵袭能力,降低MMP-9、MMP-14、VEGF表达水平,为胃癌的治疗研究提供新思路和理论基础。     

Modulation of invasive properties of human glioblastoma cells stably expressing amino-terminal fragment of urokinase-type plasminogen activator

The binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR) on the surface of tumor cells is involved in the activation of proteolytic cascades responsible for the invasiveness of those cells. The diffuse, extensive infiltration of glioblastomas into the surrounding normal brain tissue is believed to rely on modifications of the proteolysis of extracellular matrix components; blocking the interaction between uPA and uPAR might be a suitable approach for inhibiting glioma tumorigenesis. We assessed how expression of an amino-terminal fragment (ATF) of uPA that contains binding site to uPAR affects the invasiveness of SNB19 human glioblastoma cells. SNB19 cells were transfected with an expression plasmid (pcDNA3-ATF) containing a cDNA sequence of ATF-uPA. The resulting ATF-uPA-expressing clones showed markedly less cell adhesion, spreading, and clonogenicity than did control cells. Endogenous ATF expression also significantly decreased the invasive capacity of transfected glioblastoma cells in Matrigel and spheroid-rat brain cell aggregate models. ATF-uPA transfectants were also markedly less invasive than parental SNB19 cells after injection into the brains of nude mice, suggesting that competitive inhibition of the uPA-uPAR interaction on SNB19 cells by means of transfection with ATF cDNA could be a useful therapeutic strategy for inhibiting tumor progression.     

Stimulation of tumor growth by human soluble intercellular adhesion molecule-1

Because serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) are elevated in cancer and sICAM-1 is angiogenic, we tested the ability of sICAM-1 to promote tumor growth. Our preliminary experiments showed that exogenous sICAM-1 significantly stimulated the growth of human tumors in vivo. Human fibrosarcoma transfectants, which express ICAM-1, produce ICAM-1 on the cell surface and release sICAM-1 into the medium without any apparent effect on cell growth in vitro. We found that conditioned medium from sense ICAM-1 transfectants compared with mock or antisense ICAM-1 transfectants stimulates endothelial cell migration in vitro and neovascularization in the chick chorioallantoic membrane assay. Tumor cells transfected with sense constructs form faster growing tumors than mock- and antisense-transfected cells in both chick embryos and nude mice models. Serum levels of human sICAM-1 from nude mice bearing sense ICAM-1 transfectants correlate positively with tumor weight. Sense ICAM-1 transfectants are more proliferative and induce more blood vessel formation than mock and antisense transfectants in nude mice. Because expression of ICAM-1 does not affect tumor cell growth in vitro, the angiogenic activity of sICAM-1 produced by sense ICAM-1 transfectants may be involved in the stimulation of tumor growth. Therefore, sICAM-1 may perform dual functions that are essential for tumor growth: angiogenesis and escape from immune surveillance.     

Altered in vitro spreading and cytoskeletal organization in human glioma cells by downregulation of urokinase receptor

The interaction of urokinase-type plasminogen activator (uPA) with its cell-surface receptor (uPAR) is implicated in diverse biological processes such as cell migration, tissue remodeling, and tumor cell invasion. Recent studies indicated that uPAR can act as an extracellular matrix receptor during cell adhesion. Recently, we showed that transfection of the humanglioma cell line SNB19 with antisense uPAR resulted in downregulation of uPAR at both the mRNA and protein levels. In this study, we used SNB19 to determine how the presence or absence of uPAR promotes cellspreading and associated changes in cell morphology. Microscopic analysis of cell spreading revealed that antisense uPAR–transfected cells were larger, remained round, and did not spread efficiently over extracellular matrix substrate type IV collagen and fibronectin, unlike parental SNB19 cells, which were smaller and spindle shaped. Biochemical studies showed that antisense uPAR–transfected cells, in addition to not spreading, exhibited increased expression of α3β1 integrin but not α5β1 integrin. However, we could not find a change in the expression of extracellular matrix components or altered growth rate in these cells. Furthermore, despite the increased α3β1 integrin expression, antisenseuPAR–transfected cells failed to form an organized actin cytoskeleton when plated on type IV collagen or fibronectin, unlike parental SNB19 cells, which displayed an organized cytoskeleton. These findings show that the absence of uPAR in human glioma cells leads to morphological changes associated with decreased spreading and a disorganized cytoskeleton resulting in altered cell morphology, suggesting that coordinated expression of uPAR and integrin may be involved in spreading of antisense uPAR–transfected glioma cells. Mol. Carcinog. 20:355–365, 1997. © 1997 Wiley-Liss, Inc.     

Suppression of invasion and peritoneal carcinomatosis of ovarian cancer cell line by overexpression of bikunin

Bikunin (bik), a Kunitz-type protease inhibitor, also known as urinary trypsin inhibitor, is proposed as a main participant in the inhibition of tumor cell invasion and metastasis, possibly through the direct inhibition of cell-associated plasmin activity and suppression of urokinase-type plasminogen activator (uPA) mRNA expression. In the present study, we transfected the human ovarian carcinoma cell line HRA, highly invasive cells, with an expression vector harboring a cDNA encoding for human bik. Our study was designed to investigate the effect of bik overexpression and changes in tumor cell phenotype and invasiveness in the stably transfected clones. Bik gene transfection of HRA gave the following results: 1) transfection of HRA with the bik cDNA resulted in 5 variants stably expressing functional bik; 2) bik(+) clones exhibited a significantly reduced uPA mRNA expression as compared to the parental cells; 3) bikunin negatively regulates the ERK1/2 activity; 4) secretion-blocking treatments of bik(+) clones abrogated bik-mediated suppression of ERK1/2 activation and uPA expression; 5) the regulation of invasion seen in the HRA cells is mainly mediated by the uPA-plasmin-MMP-2 system; 6) transfection of HRA with the bik gene significantly reduced invasion, but not proliferation, adhesion, or migration relative to the parental cells; and 7) animals with bik(+) clones induced reduced peritoneal dissemination and long term survival. We conclude that transfection of HRA cells with the bik cDNA constitutively suppresses ERK1/2 activation, which results in inhibition of uPA expression and subsequently reduces dissemination of bik(+) clones.