Glial cell-induced endothelial morphogenesis is inhibited by interfering with extracellular signal-regulated kinase signaling.

PURPOSE: Tumor vasculature provides the infrastructure by which malignant tissue can be nourished; therefore, targeting angiogenesis may be an effective means of treating cancer. We showed previously that SNB19 glioblastoma cells modulate bovine retinal endothelial cells in cocultures to form capillary-like network structures, that matrix metalloproteinase-9 (MMP-9) expression is critical for endothelial morphogenesis, and that MMP-9 expression in glioblastoma cells is regulated by extracellular signal-regulated kinase-1 (ERK-1). In the present study, we investigated whether interfering with the activation of this mitogen-activated protein (MAP) kinase would repress MMP-9 synthesis and inhibit capillary formation. EXPERIMENTAL DESIGN: Cocultures of bovine retinal endothelial and SNB19 cells were analyzed for MMP-9 secretion, and phospho- and total ERK levels. These cocultures were treated with PD98059, a specific inhibitor of MAP/ERK kinase 1, or transfected with dominant-negative ERK-1 mutant containing expression vector. Alterations in capillary-like structure formation, and actin cytoskeleton and secretion of vascular endothelial growth factor (VEGF), MMP-9, and tissue inhibitor of metalloproteinase-1 were determined by immunofluorescence, gelatin zymography, and Western blotting. RESULTS: We found that inhibition of the ERK-1/2 pathway with PD98059 abrogated glial cell-mediated capillary formation by the endothelial cells and reduced the levels of MMP-9 in the coculture. Strikingly, the abrogation of MAP kinase signaling by a dominant-negative ERK-1 mutant inhibited glial-induced capillary network formation by reducing VEGF levels and MMP-9 activity and increasing the levels of tissue inhibitor of metalloproteinase-1. Inhibition of ERK activity also disrupted the formation of the actin cytoskeleton, a prerequisite for endothelial cell migration. CONCLUSION: The mechanism underlying activation of ERK is involved in reorganization of the actin cytoskeleton, and induction of VEGF and MMP-9, thereby stimulating endothelial cell morphogenesis. These studies clearly provide experimental evidence that ERK inhibition diminishes glial-induced endothelial-cell morphogenesis; therefore, interfering with ERK signaling may be a viable approach to target angiogenesis.     

肿瘤转移抑制基因1抑制前列腺癌细胞的增殖和侵袭能力

目的 研究肿瘤转移抑制基因1(TMSG-1)转染对高转移人前列腺癌细胞体外增殖能力和侵袭能力的影响.方法 将TMSG-1全长真核表达载体稳定转染人前列腺癌细胞高转移亚系PC-3M-1E8,G418筛选,逆转录聚合酶链反应(RT-PCR)和Western blot方法选取TMSG-1过表达阳性克隆.通过活细胞计数、二苯基溴化四氮唑蓝(MTF)比色实验、软琼脂集落形成实验检测体外细胞生长能力;Matrigel穿膜实验检测肿瘤细胞体外侵袭能力.结果 在稳定转染TMSG-1基因的PC-3M-1E8细胞系中,筛选出3株TMSG-1转录活性及蛋白表达水平均明显升高的细胞用于后续生物学行为实验.活细胞计数和MTT比色实验结果显示,从计数第3天起,与空载体对照组和空白对照组相比,3株正义转染组细胞生长速度均明显减慢(P<0.05).软琼脂集落形成实验结果显示,3株正义转染组的细胞软琼脂集落形成数与空载体对照组、空白对照组相比,均明显减少(P<0.05),分别为26.00±3.21、13.33±1.45和32.83±2.18.Matrigel穿膜实验结果显示,正义转染的各组细胞与空载体对照组及空白对照组相比,穿膜细胞数均明显减少(P<0.05),分别为45.33±4.16、54.00±2.83和26.33±3.79.结论 TMSG-1表达上调可使高转移人前列腺癌细胞体外增殖速度、锚着不依赖性生长能力及侵袭能力明显降低,TMSG-1可能通过抑制肿瘤细胞生长和侵袭抑制肿瘤转移潜能.     

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.     

Survivin基因沉默下调血管形成相关因子表达抑制视网膜母细胞瘤侵袭性及其分子机制研究

目的:研究基因沉默Survivin对视网膜母细胞瘤HXO-RB44细胞凋亡、侵袭以及血管内皮生长因子（VEGF）、基质金属蛋白酶 2（MMP-2）、基质金属蛋白酶9（MMP-9）活性的影响。方法:培养视网膜母细胞瘤HXO-RB44细胞,构建Survivin-shRNA载体。按照处理不同分为Survivin-shRNA组、GFP组和CON组。分别检测三组HXO-RB44细胞凋亡指数、细胞侵袭能力以及VEGF、MMP-2、MMP-9、CAS-3蛋白的表达水平。结果:流式细胞术结果表明,与CON组和GFP组相比,Survivin-shRNA组细胞凋亡率增加,差异有统计学意义（P＜0.05）;Transwell侵袭小室实验显示Survivin-shRNA能明显降低HXO-RB44细胞的侵袭能力;Western blot结果发现,Survivin-shRNA可降低VEGF、MMP-2、MMP-9蛋白的表达水平,并提高CAS-3的表达水平。结论:Survivin-shRNA可诱导HXO-RB44细胞凋亡,抑制细胞侵袭能力,下调血管形成相关因子VEGF、MMP-2、MMP-9的表达,进而抑制肿瘤新生血管的形成。     

PTEN基因对急性淋巴细胞白血病细胞增殖、侵袭的影响

目的:观察第10号染色体同源缺失性磷酸酶-张力蛋白同源的基因（PTEN）对急性淋巴细胞白血病细胞增殖、侵袭的影响。方法:在人急性淋巴细胞白血病细胞CEM-C1中转染PTEN过表达载体（pBP-PTEN）,同时转染对照载体（pBP）,以没有转染的细胞作为对照,RT-PCR和Western blot检测转染后细胞中PTEN的水平;MTT和集落形成试验检测细胞增殖能力;Transwell小室检测细胞侵袭和迁移能力;Western blot检测PTEN、基质金属蛋白酶-2（MMP-2）、基质金属蛋白酶-9（MMP-9）、p38丝裂原活化蛋白激酶（p38MAPK）、磷酸化的p38MAPK（p-p38MAPK）的水平。结果:转染对照载体的CEM-C1细胞中PTEN水平、细胞光密度（OD）值、克隆形成数目、侵袭数目、迁移数目及PTEN、MMP-2、MMP-9、p38MAPK、p-p38MAPK水平与未转染细胞相比没有明显变化（P＞0.05）。转染PTEN过表达载体后的CEM-C1细胞中PTEN水平和p-p38MAPK水平升高,细胞OD值、克隆形成数目、侵袭数目、迁移数目和MMP-2、MMP-9水平降低,与未转染细胞相比差异具有统计学意义（P＜0.05）。结论:PTEN抑制急性淋巴细胞白血病细胞增殖、侵袭及迁移,促进p38MAPK信号通路激活。     

Glioma cells deficient in urokinase plaminogen activator receptor expression are susceptible to tumor necrosis factor-alpha-related apoptosis-inducing ligand-induced apoptosis.

PURPOSE: The urokinase plasminogen activation system comprises the ligand urokinase plasminogen activator and the receptor urokinase plasminogen activator receptor (uPAR), which play an important role in the activation of matrix-degrading enzymes that enhance the invasion of cancer cells. Earlier studies have indicated that SNB19 glioblastoma cells expressing antisense uPAR constructs lose their invasive properties when injected in vivo. Additional observations indicated that injected antisense uPAR:SNB19 cells were being lost through apoptotic elimination. EXPERIMENTAL DESIGN: SNB19, Vector, and SNB19:asuPAR were analyzed to determine cytotoxicity of tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL), receptor expression, and underlying signaling pathways using flow cytometry, immunohistochemistry, RNase protection assay, and c-Jun-NH(2)-terminal kinase activity. RESULTS: This study elucidated the susceptibility of antisense uPAR:SNB19 cells to TRAIL under certain experimental conditions in vitro. These uPAR-deficient transfected cells had higher levels of the TRAIL receptors DR4 and DR5 than did the control and vector population as detected by flow cytometry. An RNase protection assay confirmed the elevation of DR4 and DR5 mRNA in the antisense uPAR cells. CONCLUSIONS: These findings provide preliminary evidence of a link between TRAIL-induced apoptosis and cell cycle progression in antisense uPAR:SNB 19 cells.     

Rac1基因对纤维肉瘤细胞侵袭胶原蛋白屏障的影响和机制研究

 目的　体外研究外源Rac1基因表达对HT1080纤维肉瘤细胞侵袭胶原蛋白屏障的影响及其机制。方法　将转染显性负调控突变体Rac1V12N17（HN）、持续活化型Rac1V12（HV）或空载体（HW）纤维肉瘤HT1080细胞，在含胶原蛋白凝胶三维基质中培养， 用得克萨斯红结合的鬼笔环肽染色显示细胞肌动蛋白骨架结构。用胶原蛋白凝胶薄膜覆盖滤膜的Transwell小室进行细胞侵袭胶原屏障实验，并观察2种蛋白酶抑制剂对上述细胞侵袭实验的影响。采用明胶酶谱法检测在三维基质中培养的上述转染细胞分泌型基质金属蛋白酶（MMP）-2和MMP-9的表达和活化。结果　胶原蛋白凝胶中培养的HV和HN细胞在形态上表现出明显差异，前者有更多伪足样突起；HV细胞侵袭胶原屏障能力大于HW细胞，而后者又强于HN 细胞，这种差别在应用广谱MMP抑制剂后消失，而抑肽酶则无影响；外源Rac1的表达促进胶原和纤维蛋白基质中培养的HT1080纤维肉瘤细胞分泌型MMP-2的表达和活化。结论　外源持续活化型Rac1基因在纤维肉瘤HT1080细胞内稳定表达，可诱导细胞内肌动蛋白聚集，增强细胞侵袭胶原屏障能力。Rac1表达促进MMP-2活化可能是其重要机制之一。     

TAGLN基因过表达对乳腺癌细胞迁移和侵袭能力的影响

目的 研究TAGLN基因过表达对人乳腺癌细胞MDA-MB-231迁移能力和侵袭能力的影响及其分子机制.方法 对MDA-MB-231细胞采用慢病毒表达系统构建TAGLN基因稳定过表达细胞株,将MDA-MB-231细胞正常培养设为空白对照组,空载体慢病毒包装感染MDA-MB-231细胞后获得的稳定转染细胞株设为空载体对照组.Real time PCR和Western blot检测TAGLN mRNA和蛋白表达,划痕实验和Transwell侵袭实验检测细胞迁移和侵袭能力,Western blot检测TAGLN基因过表达后基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)和MMP-9蛋白表达变化.结果 MDA-MB-231细胞感染TAGLN基因过表达慢病毒载体后,细胞中TAGLN mRNA表达和蛋白表达升高(均P＜0.01),成功构建TAGLN基因过表达稳定细胞株(231-TAGLN).231-TAGLN细胞的体外迁移能力和侵袭能力下降,与空载体对照组和空白对照组细胞比较,差异均具有统计学意义(均P＜0.01),同时伴有MMP-2和MMP-9表达水平降低(均P＜0.01).结论 TAGLN基因过表达可以抑制乳腺癌细胞的迁移和侵袭,MMP-2和MMP-9基因表达下降可能参与这一过程.     

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.     

Activation of caspase-9 with irradiation inhibits invasion and angiogenesis in SNB19 human glioma cells

Glioblastoma multiforme, the most common brain tumor, typically exhibits markedly increased angiogenesis, which is crucial for tumor growth and invasion. Antiangiogenic strategies based on disruption of the tumor microvasculature have proven effective for the treatment of experimental brain tumors. Here, we have overexpressed human caspase-9 by stable transfection in the SNB19 glioblastoma cell line, which normally expresses low levels of caspase-9. Our studies revealed that overexpression of caspase-9 coupled with radiation has a synergistic effect on the inhibition of glioma invasion as demonstrated by Matrigel assay (> 65%). Furthermore, sense caspase stable clones cocultured with fetal rat brain aggregates along with radiation showed complete inhibition as compared to the parental and vector controls. During in vitro angiogenesis, SNB19 cells cocultured with human microvascular endothelial cells (HMEC) showed vascular network formation after 48-72 h. In contrast, these capillary-like structures were inhibited when HMEC cells were cocultured with sense caspase stable SNB19 cells. This effect was further enhanced by radiation (5 Gy). Signaling mechanisms revealed that apoptosis is induced by cleavage of caspase-9 by radiation, loss of mitochondrial membrane potential and activation of caspase-3. These results demonstrate that activation of caspase-9 disrupts glioma cell invasion and angiogenesis in vitro. Hence, overexpression of proapoptotic molecules such as caspase-9 may be an important determinant of the therapeutic effect of radiation in cancer therapy.     

Down-regulation of MT1-MMP expression suppresses tumor cell invasion in metastatic human SW626 ovarian cancer cells

Membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP-14) is a key enzyme involved in degradation of extracellular matrix (ECM) and various surface-associated proteins that control cell growth, differentiation and survival, plays crucial roles in molecular carcinogenesis, tumor cell growth, invasion, and angiogenesis. We tested the inhibitory effect of antisense MT1-MMP on the ability of metastatic human ovarian carcinoma cell line SW626 in proliferation and invasion. RT-PCR was used to amplify MT1-MMP cDNA fragments with two different restriction sites at its 5'-end. Antisense MT1-MMP cloned in eukaryotic expression vector pMMP14as was transfected into SW626 cells. MT1-MMP protein expression, activities of MMP-2 and MMP-9, changes of cell proliferation, and cell invasion ability were detected by Western blot, optimized gelatin zymography, MTT assay and matrigel in vitro invasion assay, respectively. After 48 h transfection, decreased expression of endogenous MT1-MMP protein was detected in pMMP14as-transfected SW626 cells and showed significantly lower proliferation level when compared with control cells. The activation of proMMP-2 was inhibited markedly, and the mean percentage of invasive cells was 63.30+/-5.80% in pMMP14as-transfected cells, which was less than that (97.60+/-7.50%) in control cells (P<0.05). Both cell proliferation and invasion in SW626 cells were inhibited effectively by antisense MT1-MMP transfection, suggesting that MT1-MMP may be a proper target molecule for anti-invasion therapy for human ovarian cancers.     

Inhibition of cathepsin B and MMP-9 gene expression in glioblastoma cell line via RNA interference reduces tumor cell invasion, tumor growth and angiogenesis

Extracellular proteases have been shown to cooperatively influence matrix degradation and tumor cell invasion through proteolytic cascades, with individual proteases having distinct roles in tumor growth, invasion, migration and angiogenesis. Matrix metalloproteases (MMP)-9 and cathepsin B have been shown to participate in the processes of tumor growth, vascularization and invasion of gliomas. In the present study, we used a cytomegalovirus promoter-driven DNA template approach to induce hairpin RNA (hpRNA)-triggered RNA interference (RNAi) to block MMP-9 and cathepsin B gene expression with a single construct. Transfection of a plasmid vector-expressing double-stranded RNA (dsRNA) for MMP-9 and cathepsin B significantly inhibited MMP-9 and cathepsin B expression and reduced the invasive behavior of SNB19, glioblastoma cell line in Matrigel and spheroid invasion models. Downregulation of MMP-9 and cathepsin B using RNAi in SNB19 cells reduced cell-cell interaction of human microvascular endothelial cells, resulting in the disruption of capillary network formation in both in vitro and in vivo models. Direct intratumoral injections of plasmid DNA expressing hpRNA for MMP-9 and cathepsin B significantly inhibited established glioma tumor growth and invasion in intracranial tumors in vivo. Further intraperitoneal (i.p.) injections of plasmid DNA expressing hpRNA for MMP-9 and cathepsin B completely regressed pre-established tumors for a long time (4 months) without any indication of these tumor cells. For the first time, these observations demonstrate that the simultaneous RNAi-mediated targeting of MMP-9 and cathepsin B has potential application for the treatment of human gliomas.     

Overexpression of vimentin: role in the invasive phenotype in an androgen-independent model of prostate cancer

The androgen-sensitive LNCaP prostate cancer cell line is less invasive than hormone-insensitive lines. CL1, an aggressive, hormone-insensitive LNCaP subline derived by continuous passaging in hormone-depleted medium, was compared with the parental cell line by cDNA microarray analysis. The gene coding for the intermediate filament protein vimentin was found to be highly up-regulated in the CL1 subline. This difference was confirmed by Northern and Western blots and visualized by immunofluorescence microscopy. To assess the contribution of vimentin to the invasive phenotype, LNCaP cells were stably transfected to overexpress vimentin, and the CL1 cells were transfected with vimentin antisense construct. The invasiveness of the transfected cells was tested using an in vitro invasion assay. We were able to demonstrate that decreasing vimentin expression in the constitutively vimentin-expressing CL1 cells led to a significant decrease in their invasiveness but that forcing expression of vimentin in the LNCaP cells did not augment their invasiveness. These findings imply that vimentin expression contributes to the invasive phenotype but cannot confer it alone.