双调蛋白反义核酸表达对裸鼠乳腺癌血管生成的抑制作用

目的 研究双调蛋白（AR）反义RNA表达对乳腺癌血管生成的抑制作用。方法 乳腺癌NS2T2A1细胞经AR反义cDNA质粒转染后,经筛选获得表达AR反义RNA的AR-AS1及AR-AS3克隆,转染空载体获得NS2T2A1 V对照细胞,接种裸鼠皮下形成肿瘤。研究条件培养液对人微血管内皮细胞（HMEC）增殖的影响,以EL1SA法测定细胞血管内皮生长因子（VEGF）分泌量。以定量RT-PCR分析肿瘤组织的VEGF表达水平。以免疫组化法标记CD31研究肿瘤内血管数量。结果 HMEC在AR-AS1和AR-AS3细胞条件培养液中的增殖比例明显降低。AR-AS1和AR-AS3细胞VEGF分泌量亦降低。AR-AS1和AR-AS3肿瘤的血管数量仅有对照组的50％左右,且VEGF表达显著降低。结论 AR反义RNA表达可有效抑制乳腺癌的血管生成,应作为新治疗靶点进一步研究．     

双调蛋白反义重组腺病毒治疗乳腺癌的实验研究

目的:在人乳腺癌模型上探讨双调蛋白(AR)反义重组腺病毒的治疗作用.方法:人AR的cDNA被反向亚克隆入E1/E3缺失的5型腺病毒载体中而得到AdA4,AR反义mRNA的表达由CMV启动子控制.进而研究其对恶性转化人乳腺上皮细胞株NS2T2A1体外、体内增殖的作用.结果:乳腺癌NS2T2A1细胞被AR反义重组腺病毒感染后,AR蛋白质的表达被强烈地抑制.以MOI为200和400pfu/细胞的AdA4感染4天后,细胞体外增殖分别被显著地抑制至空病毒对照组的69.3%和49.8%(P＜0.02,P＜0.005).3次肿瘤内注射109pfu AdA4病毒,至第35天肿瘤体积只为对照组的40.6%(P＜0.005).结论:在此乳腺癌模型上,AR反义重组腺病毒可有效地用于针对AR的反义治疗策略,抑制肿瘤细胞的体外增殖及体内肿瘤生长.     

结缔组织生长因子转染对人乳腺癌细胞系生物学行为的影响

背景与目的:结缔组织生长因子(CTGF)是CCN家族成员之一,参与体内多种生理和病理生理过程。本研究探讨CTGF对人乳腺癌细胞生物学行为的影响。方法:构建含有CTGF开放读码框的真核表达质粒,通过脂质体介导的方法将正义质粒转染MCF-7细胞,反义质粒转染MDA-MB-231细胞。观察CTGF不同表达水平与乳腺癌细胞增殖、凋亡、周期分布和体外侵袭等生物学行为的关系。结果:转染正义质粒使MCF-7细胞CTGF表达上调,抑制细胞增殖,促进细胞凋亡,细胞发生G0G1期阻滞,体外侵袭能力下降。转染反义质粒使MDA-MB-231细胞CTGF表达下调,促进细胞增殖,减少细胞凋亡,增强细胞体外侵袭能力,但对细胞周期无影响。结论:CTGF具有抑制乳腺癌细胞生长和侵袭的功能。促进细胞凋亡是CTGF抑制乳腺癌细胞体外生长的机制之一。CTGF可能对细胞周期有一定的影响。     

MTA1沉默对膀胱癌细胞株BIU-87侵袭能力影响的探讨

目的:采用RNAi沉默膀胱癌细胞株BIU-87 MTA1基因的表达,观察对膀胱癌细胞侵袭能力的影响。方法:构建针对MTA1的siRNA表达质粒,体外转染BIU-87细胞。分别采用RT-PCR和蛋白质印迹法检测各组MTA1 mRNA和蛋白表达;Milli-cell PCF侵袭小室及肿瘤细胞体外黏附试验研究转染前后膀胱癌细胞侵袭能力的变化。结果:与转染空质粒组和未转染组相比,转染siRNA载体组细胞MTA1表达在RNA及蛋白水平都显著低于对照组,前组穿透侵袭小室滤膜的数目明显少于空质粒组和未转染组(F=213.147,P=0.000),MTT法测定已黏附细胞的OD490 nm,显示前组的体外黏附能力与转染空质粒组和未转染组体外黏附能力差异有统计学意义,F=5.663,P=0.008。结论:shMTA1可抑制MTA1在膀胱癌细胞中的表达,并可抑制膀胱癌细胞侵袭转移,以MTA1为靶点的RNA干扰技术可望成为膀胱癌基因治疗的新方法。     

MT1-MMP反义核酸抑制高转移人卵巢癌SW626细胞的侵袭效应

背景与目的:MT1-MMP(MMP-14)是新发现的一种膜型基质金属蛋白酶,研究表明MT1-MMP在肿瘤转移中发挥关键性作用。本研究应用反义核酸技术,观察了MT1-MMP反义核酸对高转移人卵巢癌SW626细胞体外生长和侵袭的抑制效应。方法:应用自行设计的引物,借助RT-PCR技术获得两端含不同限制酶位点的MT1-MMPcDNA片段,反向插入pcDNA3.1中,并应用脂质体介导的基因转染技术,将重组质粒导入SW626细胞中,应用MTT、Westernblot、明胶酶谱和体外侵袭实验等方法观察了SW626细胞转染前后,细胞生长、MT1-MMP蛋白表达、MMP-2和MMP-9酶活性及细胞体外侵袭能力等指标的变化。结果:成功构建了反义MT1-MMP真核表达载体(pMMP14as),将其转染入SW626细胞后,与空质粒转染组相比,MT1-MMP反义核酸转染组细胞MT1-MMP表达显著降低,抑制率为65.8%;MMP-2酶原的活化受到明显抑制;pMMP14as转染组的穿膜细胞百分数为(63.3±5.8)%,明显低于空质粒转染组(97.6±7.5)(P<0.05)%。结论:MT1-MMP反义核酸可明显抑制高转移SW626细胞体外生长和侵袭效应,提示MT1-MMP可作为人卵巢癌抗侵袭治疗的分子靶点。     

VEGF-C反义寡核苷酸对乳腺癌细胞MDA-MB-435黏附、侵袭能力的影响

目的　探讨 VEGF- C反义寡核苷酸 (antisense oligodeoxynucleotide,ASODN )对人乳腺癌细胞 MDA-MB- 4 35黏附、侵袭能力的影响。方法　以 VEGF- C基因编码区为靶点合成反义寡核苷酸 ,脂质体介导转染。 RT-PCR法检测转染后 VEGF- C m RNA表达水平 ;MTT法检测乳腺癌细胞与细胞外基质黏附 ;利用 Transwell小室 ,检测乳腺癌细胞侵袭人工基底膜能力 ;Western- blot法检测乳腺癌细胞中 MMP- 2蛋白表达。结果　与空白对照及正义序列 (sense oligodeoxynucleotide,SODN)转染组比较 ,ASODN组 VEGF- C m RNA水平下调 ,差异有显著性 (t=5 .6 4 ,5 .4 8,P<0 .0 1) ;对胞外基质黏附率明显降低 (χ2 =7.15 ,7.2 9,P<0 .0 5 ) ,穿透重组基底膜数目显著下降 (t=5 .2 9,5 .17,P<0 .0 1) ;细胞裂解液中 MMP- 2蛋白表达显著下调 (t=7.36 ,7.4 3,P<0 .0 1)。结论　 VEGF- C基因 ASODN脂质体介导转染可明显抑制乳腺癌细胞体外实验中的黏附和侵袭能力 ;下调 MMP- 2的表达可能是其作用途径。     

Cathepsin B反义RNA抑制乳腺癌细胞的侵袭和迁移

摘 要　目的：探讨人cathepsin B(CatB)反义RNA在乳腺癌细胞侵袭与迁移中的作用。方法: 构建携CatB反义RNA的重组质粒pBudCE4.1-antiCatB,采用脂质体法将重组质粒瞬时转染人乳腺癌细胞MDA-MB-231。Western blotting法检测MDA-MB-231细胞中CatB蛋白的表达, MTT法检测MDA-MB-231细胞的增殖,细胞-基质黏附实验检测反义CatB对MDA-MB-231细胞黏附能力的影响,体外侵袭、迁移实验分析反义CatB表达对MDA-MB-231细胞体外侵袭和迁移能力的影响。结果:成功构建pBudCE4.1-antiCatB表达载体。转染MDA-MB-231细胞后,与未转染组细胞和转染空载体组细胞相比,转染组MDA-MB-231细胞的CatB蛋白表达水平明显降低[(0.96±0.02) vs(1.98±0.23),(1.84±0.08),P<0.05];转染组细胞的增殖受到明显抑制[(0.255±0.017) vs(0.458±0.033),(0.421±0.022),P<0.01];转染组细胞的黏附（基质胶或纤维黏连蛋白）能力明显下降[(0.054±0.017) vs(0.111±0.018),(0.107±0.017), P<0.01;或(0.052±0.008) vs(0.120±0.014),(0.113±0.009), P<0.01];转染组细胞的侵袭和迁移能力也明显降低[(52.80±7.76) vs(124.00±44.54),(116.80±32.87), P<0.01;(60.25±8.73) vs(132.50±12.15),(119.20±25.13), P<0.01]。结论:CatB反义RNA可抑制乳腺癌细胞体外生长、黏附、迁移和侵袭能力。     

血管内皮生长因子反义cDNA对人肺癌抑瘤作用的体外实验研究

目的：研究血管内皮生长因子(VEGF)反义cDNA对肿瘤细胞VEGF mRNA和蛋白表达的影响，并探讨其对肿瘤细胞生长、增殖过程的作用。方法：以脂质体介导VEGF反义cDNA转染肺癌细胞，通过原位杂交法检测细胞VEGF mRNA．免疫组化法检测细胞VEGF蛋白和PCNA增殖活性，ELISA法检测分泌型VEGF蛋白，MTT法测定细胞生长率．研究VEGF反义cDNA对肿瘤细胞的作用。结果：转染后肺癌细胞内源性VEGF mRNA和蛋白的表达受抑．细胞生长及增殖受抑。结论：VEGF反义cDNA转染可降调节肿瘤细胞的VEGF生成并抑制其生长与增殖．     

外源性AKT1增强上皮性卵巢癌细胞侵袭和迁移能力的机制

探讨AKT1基因对上皮性卵巢癌SKOV3细胞迁移和侵袭能力的影响及相关分子机制。方法 针对AKT1基因,设计并构建shRNA质粒和真核表达质粒,双向调节SKOV3细胞中AKT1的表达,运用RT-PCR和western blot检测转染效率。运用Wound healing和Transwell-Matrigel方法检测转染前后细胞迁移和侵袭能力的变化。RT-PCR法检测与细胞运动侵袭相关分子CXCR4、VEGF、MMP-2、MMP-9和uPA在mRNA水平的表达变化。结果 成功构建AKT1基因的真核表达质粒pEF-1α-AKT1和靶向抑制AKT1基因的shRNA表达质粒pRNAT-AKT1。转染上皮性卵巢癌SKOV3细胞后,能有效调控p-AKT表达。参照未转染组和空载体转染组,外源性AKT1促进细胞迁移和侵袭,CXCR4、VEGF、MMP-2和uPA的mRNA表达水平升高。shRNA靶向抑制AKT1基因的表达可抑制细胞迁移和侵袭,CXCR4、VEGF、MMP-2和uPA的mRNA表达水平下降。结论 AKT1可能通过调控CXCR4、VEGF、MMP-2和uPA的转录水平来影响细胞侵袭和运动能力。     

转染缝隙连接蛋白CX43在脑胶质瘤化疗旁观者效应影响的体外实验研究

背景与目的:肿瘤细胞受损伤时,其旁边未受损肿瘤细胞也出现损伤的现象称为旁观者效应,国内外研究发现多种肿瘤组织在体内外存在该效应,为进一步明确脑胶质瘤在药物化疗时是否存的旁观者效应以及CX43(缝隙连接蛋白43)对该效应的影响,通过构建并转染pCMV-Cx43cDNA质粒入C6细胞,上调缝隙连接蛋白CX43表达,增强缝隙连接功能,探讨转染缝隙连接蛋白CX43对脑胶质瘤化疗体外旁观者效应影响。方法:(1)通过C6细胞培养,并提取总RNA,行RT-PCR、质粒酶切及连接等,构建缝隙连接蛋白CX43真核表达重组质粒pCMV-Cx43cDNA;(2)通过脂质体行重组质粒pCMV-Cx43cDNA转染C6细胞,过表达C6细胞缝隙连接蛋白CX43,RT-PCR及Weston-blot检测CX43 mRNA及蛋白表达水平变化。划痕荷载染料传输实验法检测转染缝隙连接蛋白CX43后缝隙连接功能;(3)通过体外TRASWELL细胞共培养模型,将转染pCMV-Cx43cDNA、空载体细胞分别分成化疗药物替膜唑胺处理组与替膜唑胺未处理组细胞,然后将两组细胞按一定比例分别接种TRASWELL细胞共培养板膜两侧,检测未化疗处理细胞存活率及凋亡率,观察体外转染CX43对体外化疗旁观者影响。结果:(1)成功构建带CX43目的基因的真核表达pCMV-Cx43cDNA重组质粒;(2)成功转染C6细胞并筛选稳定转染过表达CX43细胞株(C6-Cx43)及转染空载体的细胞株(C6-non),C6-Cx43细胞Cx43mRNA及CX43蛋白表达显著增加;(3)在体外TRASWELL细胞共培养模型中,观察到体外未化疗细胞凋亡的旁观者效应,(C6-Cx43)组与对照组差异存在明显统计学意义。结论:(1)C6细胞转染重组质粒pCMV-Cx43cDNA,缝隙连接蛋白CX43表达增高,C6细胞GJIC功能增强;(2)在体外化疗时,C6-Non、C6-Cx43都存在化疗旁观者效应,而C6-Cx43的化疗旁观者效应显著。即转染缝隙连接蛋白CX43能放大体外化疗的旁观者效应。     

Antisense expression for amphiregulin suppresses tumorigenicity of a transformed human breast epithelial cell line

The epidermal growth factor (EFG) family of receptors and their respective ligands play a major role in breast cancer progression and are the targets of new therapeutic approaches. Following immortalization with SV40 T antigen of normal human breast epithelial cells, a transformed variant cell line (NS2T2A1) was selected for its increased tumorigenicity in nude mice. This cell line was shown to have a higher expression of EGF receptors (EGFR) and amphiregulin (AR) when compared to their normal counterparts or less aggressive transformed cells. Dual staining of EGFR and AR was observed in 50-60% of NS2T2A1 cells, while 30-40% cells expressed AR only. To explore the potential tumorigenic role of AR, a 1.1 kb AR cDNA in an antisense orientation was transfected in NS2T2A1 cells. Three clones, selected by hygromycin B, expressed AR antisense RNA (AR AS1, AR AS2 and AR AS3 cell lines) in which AR protein expression was reduced (ranging from about 50 to < 5%). The anchorage-independent growth of AR AS cell lines was reduced to levels ranging from 32.4-6.8% relative to the control cell line transfected with the vector alone. The clones expressing AR antisense RNA showed a reversion of the malignant phenotype when injected in nude mice, since a significant reduction of tumor intake was observed coincident with a significant tumor mass reduction (> 96%). Moreover, intra-tumoral vascularization decreased significantly in tumors derived from AR AS cells (26.7, 70.7 and 50.4% of control). These in vitro and in vivo data reveal the oncogenic nature of AR in transformed breast epithelial cells and imply a role for AR in tumor angiogenesis.     

Amphiregulin Antisense RNA Delivered by Adnovirus Suppresses Growth of Breast Cancer Cells In Vitro and In Vivo

OBJECTIVE To investigate the therapeutic potential of amphiregulin antisense RNA delivered by adenoviral vector in a human breast cancer model.METHODS Human amphiregulin cDNA was subcloned in the opposite orientation to the cytomegaloviral promoter and inserted into an E1/E3-deleted type 5 adenoviral vector to obtain an AdA4 construct which expresses amphiregulin antisense mRNA. Both in vitro and in vivo antiproliferative effects of the antisense RNA were studied by infecting transformed human breast epithelial NS2T2A1 cells and tumors.RESULTS Amphiregulin protein expression was inhibited dramatically in the NS2T2A1 cells after infection with AdA4. The in vitro cell growth was inhibited significantly at day 4 post-AdA4 infection compared with control empty virus AdC1 at a MOl of 200 and 400 pfu/cell to 69.3% and 49.8%, respectively (P<0.02, P<0.005). After 3 intra-tumoral injections of 109 pfu AdA4, tumor volumes were reduced to 40.6% of that of the control group at day 35 (P<0.005).CONCLUSION The transfer of amphiregulin RNA antisense by adenoviral vector is effective for amphiregulin targeting strategy, leading to an inhibition of in vitro cell proliferation and in vivo tumor growth in this breast cancer model.     

Regulation of extracellular matrix metalloproteinase inducer and matrix metalloproteinase expression by amphiregulin in transformed human breast epithelial cells

Amphiregulin (AR) and epidermal growth factor effects on expression and activity of the extracellular matrix metalloproteinase inducer (EMMPRIN) were examined in NS2T2A1 breast tumor cells. Both growth factors induced mRNA and protein expression of EMMPRIN, and matrix metalloproteinase (MMP) -2 and -9 enzymatic activity. The induction of EMMPRIN by AR was mediated by epidermal growth factor receptor (EGFR) tyrosine kinase activation and inhibited by ZD1839. AR and EGFR antisense (AS) cDNAs inhibited EMMPRIN expression and MMP activity. Coculture of NS2T2A1V expressing AR- or EGFR-AS with fibroblasts and endothelial cells showed a decreased MMP activity. In parallel, nude mice tumors derived from AR and EGFR-AS cells revealed reduced level of EMMPRIN and MMP activity. AR and epidermal growth factor, therefore, regulate EMMPRIN and its MMP-mediated expression, identifying EGFR signaling as critical to this regulation.     

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.     

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.     

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.     

Her-2/neu and urokinase-type plasminogen activator and its inhibitor in breast cancer.

PURPOSE: Recent studies suggest that HER-2/neu specifically promotes the invasive capacity of tumor cells by up-regulating secretion of the proteolytic enzyme, urokinase-type plasminogen activator (uPA), or its inhibitor, plasminogen activator inhibitor-1 (PAI-1), in colon and gastric cancer. It was the purpose of this study to: (a) evaluate the association between HER-2/neu and uPA and PAI-1 expression in a large primary breast cancer cohort; (b) perform the first multivariate analysis, including HER-2/neu, uPA, and PAI-1 in breast cancer; and (c) define the effect of HER-2/neu overexpression on uPA and PAI-1 expression in breast cancer cells. EXPERIMENTAL DESIGN: HER-2/neu, uPA, and PAI-1 were measured as continuous variables by ELISA in primary breast cancer tissue extracts from 587 patients with clinical follow-up and analyzed for correlations with clinical outcome. Furthermore, a full-length human HER-2/neu cDNA was introduced into five human breast cancer cell lines to define the effects of HER-2/neu overexpression on uPA and PAI-1 expression. In addition, we tested whether HER-2/neu antibodies could reverse any given alteration of uPA and PAI-1 levels. RESULTS: Our findings indicate a weak positive association between HER-2/neu and uPA (r = 0.147; P < 0.001) and no association between HER-2/neu and PAI-1 (r = 0.07; P = 0.085). HER-2/neu overexpression (> or =400 fmol/mg) and high levels of uPA/PAI-1 (> or =5.5 ng/mg and/or > or =14 ng/mg, respectively) were significantly associated with shorter disease-free survival (DFS; P < 0.001 and P = 0.003) and metastasis-free survival (MFS; P = 0.015 and P < 0.001). Multivariate analysis revealed prognostic independence between HER-2/neu and the uPA/PAI-1 axis for DFS and MFS. Both uPA and PAI-1 had no significant discriminatory effect among HER-2/neu-positive patients for DFS. The prognostic value of HER-2/neu overexpression for MFS, however, was significantly enhanced by elevated uPA expression (P = 0.053). Stable transfection of the HER-2/neu gene into multiple human breast cancer cell lines resulted in consistent down-regulation of uPA or PAI-1 expression. In addition, anti-HER-2/neu antibodies did not significantly affect uPA or PAI-1 expression in human cancer cell lines naturally overexpressing HER-2/neu. CONCLUSIONS: The present findings suggest that the invasive phenotype elicited by HER-2/neu overexpression in breast cancer is not a direct effect of uPA or PAI-1 expression. HER-2/neu and the uPA/PAI-1 axis have been shown to affect the invasive capacity of breast cancer independently. Determination of uPA can provide significant additional prognostic information for MFS in HER-2/neu-positive and -negative patients.