Downregulation of laminin alpha4 chain expression inhibits glioma invasion in vitro and in vivo

The laminin family is a structural constituent of the extracellular matrix that plays an essential role in promoting the motility of infiltrative tumor cells. We investigated the role of laminin alpha4 chain, a subset of laminin-8, -9 and -14, in the motile and invasive activities of human glioma cells. All malignant glioma cell lines examined expressed more mRNA for the laminin alpha4 and beta1 chains than for the beta2 chain, indicating that these cells predominantly express the laminin-8 isoform. Introducing an antisense oligonucleotide for laminin alpha4 chain (AS-Ln-alpha4) into the glioma cells resulted in downregulation of laminin alpha4 expression. AS-Ln-alpha4 also significantly suppressed glioma cell adhesion and migration. Furthermore, invasiveness was significantly reduced in cells transfected with AS-Ln-alpha4 compared to those transfected with the sense oligonucleotide (S-Ln-alpha4). Indeed, when glioma spheroids were implanted into rat brain slices, AS-Ln-alpha4-transfected cells failed to invade surrounding normal brain tissues. In addition, intracerebral injection of glioma cells transfected with AS-Ln-alpha4 into nude mice resulted in the formation of a noninvasive tumor, whereas injection of cells transfected with S-Ln-alpha4 resulted in diffuse invasion of brain tissue. These results suggest that mainly laminin-8 is essential for the invasive activity of human glioma cells; thus, a novel therapeutic strategy could target this molecule to treat patients with malignant glioma.     

Adenovirus-mediated expression of antisense MMP-9 in glioma cells inhibits tumor growth and invasion

Matrix metalloproteinase 9 (MMP-9) is known to play a major role in cell migration and invasion in both physiological and pathological processes. Our previous work has shown that increased MMP-9 levels are associated with human glioma tumor progression. In this study, we evaluated the ability of an adenovirus containing a 528 bp cDNA sequence in antisense orientation to the 5' end of the human MMP-9 gene (Ad-MMP-9AS) to inhibit the invasiveness and migratory capacity of the human glioblastoma cell line SBN19 in in vitro and in vivo models. Infection of glioma cells with Ad-MMP-9AS reduced MMP-9 enzyme activity by approximately 90% compared with mock- or Ad-CMV-infected cells. Migration and invasion of glioblastoma cells infected with Ad-MMP-9AS were significantly inhibited relative to Ad-CMV-infected controls in spheroid and Matrigel assays. Intracranial injections of SNB19 cells infected with Ad-MMP-9AS did not produce tumors in nude mice. However, injecting the Ad-MMP-9AS construct into subcutaneous U87MG tumors in nude mice caused regression of tumor growth. These results support the theory that adenoviral-mediated delivery of the MMP-9 gene in the antisense orientation has therapeutic potential for treating gliomas.     

特异性下调CXCR4表达对食管鳞癌细胞迁移侵袭能力的影响

目的:探讨特异性下调CXCR4分子的表达对食管鳞癌细胞系TE-1迁移侵袭能力的影响。方法:根据CXCR4基因序列,设计合成CXCR4特异性小干扰RNA,采用siRNA技术下调CXCR4的表达,转染48h后,RT-PCR和Western blot检测各组细胞CXCR4在mRNA水平和蛋白质水平表达,通过Traswell小室实验检测细胞系迁移侵袭能力的差异。结果:小干扰RNA下调CXCR4表达后,Western blot和RT-PCR证实TE-1细胞中CXCR4表达明显下调,Traswell小室实验证实,下调CXCR4表达后,TE-1细胞的体外迁移侵袭潜能显著降低。结论:小干扰RNA下调CXCR4基因后,降低了食管鳞癌细胞TE-1的迁移侵袭能力,CXCR4表达水平与食管鳞癌的迁移侵袭能力呈正相关,抑制CXCR4表达能够使食管鳞癌细胞迁移侵袭能力明显降低。     

Rictor regulates MMP-9 activity and invasion through Raf-1-MEK-ERK signaling pathway in glioma cells

Glioblastoma multiforme (GBM) is the most common and highly aggressive type of primary brain tumor. Tumor-associated macrophages (TAMs) secrete TNF-α that activates important survival pathways including Akt (PKB)/mTOR network. The mammalian target of rapamycin (mTOR) network functions downstream of PI3K/Akt pathway to regulate cell growth, proliferation and survival. mTOR exists in two distinct complexes-mTORC1 and mTORC2 that differ in their components and sensitivity to rapamycin. The rapamycin-insensitive complex (mTORC2) consists of mTOR, mLST8, Rictor, mSin1 and Protor and regulates the actin cytoskeleton in addition to activating Akt (protein kinase B). The present study aimed to investigate the role of Rictor-a core component of mTORC2 in regulating proliferation, survival, and invasion in gliomas. siRNA-mediated loss of Rictor function in human glioma cell lines, LN18 and LN229 and in primary GBM cells resulted in elevated expression and activity of MMP-9 and significant increase in the invasive potential of these cells. Mechanistic studies revealed that the activation of Raf-1-MEK-ERK pathway was essential for induction of MMP-9 activity and enhanced invasion. Interestingly, ablation of Rictor did not affect TNF-α-induced MMP-9 activity and invasiveness suggesting that TNF-α in the microenvironment of tumor might overrule the function of Rictor as a negative regulator of MMP-9 and invasion. Silencing Rictor had no effect on the survival or proliferation in the cell lines in the presence or absence of TNF-α. Our findings identify a role for Rictor in bridging two major pathways-Akt (PKB)/mTOR and Raf-1-MEK-ERK in regulating MMP-9 activity and invasion of glioma tumor cells.     

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.     

Downregulation of heparanase by RNA interference inhibits invasion and tumorigenesis of hepatocellular cancer cells in vitro and in vivo

Heparanase is an endoglycosidase that degrades heparan sulfate, the main polysaccharide constituent of the extracellular matrix and basement membrane. The expression of heparanase is associated with invasion, as well as the angiogenic and metastatic potential of diverse malignant tumors. We used RNA interference strategies to evaluate the role of human heparanase in a liver cancer cell line and to explore the therapeutic potential of its specific targeting. Using an online siRNA tool, we designed three small interfering RNA sequences to target the heparanase coding region and cloned them into the pGenesil-1 vector. The siRNA vectors were transfected into HepG2 liver cancer cells. Heparanase expression was measured by real-time RT-PCR and Western blotting. Cell proliferation was detected by MTT staining and plate colony formation. Cell cycle analysis was performed by flow cytometry. In vitro invasion was measured by Matrigel invasion assay. We also analyzed tumorigenicity in heparanase-suppressed HepG2 cells in nude mice. We found that siRNA-1 (1214-1232) and siRNA-3 (611-629) targeting heparanase significantly downregulated the expression of heparanase in HepG2 liver cancer cells. Compared with its controls, siRNA-1 or siRNA-3 vectors efficiently inhibi-ted the proliferation and invasion of HepG2 liver cancer cells in?vitro and tumorigenesis in vivo. These results suggest that heparanase-specific RNA interference has potential value as a novel therapeutic agent for human liver cancer.     

Suppression of MMP-9 activity by NDRG2 expression inhibits clear cell renal cell carcinoma invasion

Members of the NDRG (N-Myc downstream-regulated) gene family have been shown to play a variety of roles in human malignancies. Recently, it was shown decreased expression in clear cell renal cell carcinoma (CCRCC) and inhibited cell proliferation, but the role of the NDRG2 in CCRCC invasion has not been described. We examined the expression of NDRG2 protein in CCRCC samples and the association between NDRG2 expression and CCRCC patients survival. Real-time RT-PCR and immunohistochemical analysis were used to measure NDRG2 expression in 60 paired CCRCC and adjacent normal tissues. Changes in cell invasion were detected by up- or down-regulating NDRG2 by adenovirus or siRNA. We found that NDRG2 expression is significantly down-regulated in CCRCC at mRNA and protein levels in a manner negatively associated with aggressive tumor behaviors, such as TNM stage (P?=?0.003), Fuhrman??s grade (P?=?0.024), tumor invasion (P?=?0.001) and tumor recurrence (P?=?0.004), as well as shorter patient survival rates (P?=?0.0041). Furthermore, NDRG2 could suppress CCRCC cell invasion through regulating MMP-9 expression and activity. So, these results suggest that NDRG2 can inhibit extracellular matrix-based tumor cell invasion and thereby play important roles in suppressing tumor metastasis in CCRCC. NDRG2 expression may also be a significant prognostic indicator for CCRCC.     

MMP-9和Ezrin在胶质瘤中的表达及相关性分析

目的：检测脑胶质瘤组织中MMP-9和Ezrin表达情况并探讨其意义.方法：采用免疫组织化学SP法检测50例脑胶质瘤、10例正常脑组织中两者的表达.结果：脑胶质瘤组织中MMP-9和Ezrin表达定位于细胞浆,其阳性表达率分别为74.00％ （37/50）和60.00％ （30/50）;正常脑组织为0、10.0％ （P ＜0.05）.两者的表达与胶质瘤患者的年龄、性别无明显相关性（P＞0.05）,但高级别组与低级别组之间表达具有统计学差异（P＜0.05）.在脑胶质瘤组织中MMP-9蛋白的阳性表达与Ezrin蛋白的阳性表达有相关性,二者呈正相关（P＜0.05）.结论：MMP-9高表达可能与胶质瘤发生有关,且与胶质瘤的浸润、发展、转移有关,Ezrin基因突变或缺失在胶质瘤的发生发展中起重要作用.     

Triptolide inhibits proliferation and invasion of malignant glioma cells

Malignant glioma is the most devastating and aggressive tumor in brain, characterized by rapid proliferation and diffuse invasion. Chemotherapy and radiotherapy are the pivotal strategies after surgery; however, high drug resistance of malignant glioma and the blood-brain barrier usually render chemotherapy drugs ineffective. Here, we find that triptolide, a small molecule with high lipid solubility, is capable of inhibiting proliferation and invasion of malignant glioma cells effectively. In both investigated malignant glioma cell lines, triptolide repressed cell proliferation via inducing cell cycle arrest in G0/G1 phase, associated with downregulation of G0/G1 cell cycle regulators cyclin D1, CDK4, and CDK6 followed by reduced phosphorylation of retinoblastoma protein (Rb). In addition, triptolide induced morphological change of C6 cells through downregulation of protein expression of MAP-2 and inhibition of activities of GTPases Cdc42 and Rac1/2/3, thus significantly suppressing migratory and invasive capacity. Moreover, in an in vivo tumor model, triptolide delayed growth of malignant glioma xenografts. These findings suggest an important inhibitory action of triptolide on proliferation and invasion of malignant glioma, and encourage triptolide as a candidate for glioma therapy.     

Ganglioside GM3 inhibits proliferation and invasion of glioma

GM3, the simplest ganglioside, modulates cell adhesion, proliferation and differentiation in the central nervous system and exogenously added GM3 regulates cell–cell and cell–extracellular matrix adhesion and induces apoptosis. To assess the anti-tumor action of exogenous GM3, we examined its effect on the proliferation and invasion of glioma cells. Its inhibitory effect on cell proliferation was demonstrated in vitro by 3-(4,5-dimethyl-2-thiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay and in vitro in rats with meningeal gliomatosis whose survival was significantly prolonged by the intrathecal injection of GM3. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay revealed that GM3 induced glioma cell apoptosis in vitro and in vitro. In rat brain slice cultures, GM3 suppressed the invasion of glioma cells; this effect manifested earlier than the inhibition of cell proliferation and before apoptosis induction. Our results suggest exogenous GM3 as a potential therapeutic agent in patients with glioma requiring adjuvant therapy.     

Downregulation of PAK5 inhibits glioma cell migration and invasion potentially through the PAK5-Egr1-MMP2 signaling pathway

PAK5 (p21 activated kinase 5) is upregulated in human colorectal carcinoma cells and is a known tumor promoter in carcinogenesis of the colon. Little is known regarding the mechanisms underlying the downstream targets of PAK5, and information concerning its biological significance in glioma is lacking. In this study, we investigated the effects of PAK5 on proliferation, migration, invasion, and apoptosis in human U87 and U251 glioma cells and examined the underlying molecular mechanism. We performed cell growth assays and cell cycle analysis to observe the cell proliferation. Flow cytometry analysis was performed to evaluate apoptosis, and in vitro scratch assays, cell migration assays, and gelatin zymography were performed to examine cell migration. Western blot analysis was performed to examine signal transduction in the cells. We demonstrated that suppression of PAK5 in glioma cells significantly inhibited cell migration and invasion. We also observed that suppression of PAK5 in human glioma cell lines inhibited cell growth because of G1 phase arrest. Additionally, flow cytometry and Western blot analysis indicated that PAK5 could inhibit cell apoptosis. These results suggest that the PAK5–Egr1–MMP2 signaling pathway is involved in tumor progression and may have a potential role in cancer prevention and treatment.     

Downregulation of gelsolin family proteins counteracts cancer cell invasion in vitro

Gelsolin and CapG are both actin binding proteins that modulate a variety of physiological processes by interacting differently with the actin cytoskeleton. Several studies suggest that overexpression of these proteins promotes invasion in vitro. In this study we explored the contribution of these proteins in human cancer cell invasion and motility. We show that down regulation of CapG or gelsolin in several types of cancer cells, including MDA-MB 231 and PC-3 cells, significantly reduces the invasive and motile properties of cells, as well as cell aggregation. These results point to a role for CapG and gelsolin as tumor activator.     

Downregulation of uPARAP mediates cytoskeletal rearrangements and decreases invasion and migration properties in glioma cells

To identify molecular therapeutic targets for glioma, we performed gene expression profiling by using a complementary DNA (cDNA) microarray method and identified the urokinase plasminogen activator receptor-associated protein (uPARAP/Endo180) as a gene expressed highly in glioma tissue compared with the normal brain tissue. The uPARAP is an endocytic receptor for collagen. In certain cell types, uPARAP occurs in a complex with the urokinase plasminogen activator receptor (uPAR) where it fulfills other functions in addition to collagenolysis. Quantitative PCR analysis using a cDNA panel revealed higher expression levels of uPARAP in glioma tissue compared with normal brain tissue. Western blot analysis revealed that the uPARAP protein was expressed in glioma samples and two glioma cell lines, KNS42 and KNS81, but not expressed in control tissue from the normal brain. Introduction of small interfering RNA-targeted uPARAP into the two different glioma cell lines, KNS42 and KNS81, resulted in downregulation of uPARAP expression, and it significantly suppressed glioma cell migration and invasion in vitro. Control glioma cells showed small cell bodies, whereas uPARAP siRNA-treated glioma cells exhibited large and flat morphology. Most of the polymeric actin in the control glioma cells was concentrated in the lamellipodia that are observed in mobile cells. In contrast, in the uPARAP siRNA-treated glioma cells, polymeric actin became organized in stress fibers and the lamellipodia disappeared, characteristic of immobile cells. Our present study suggests that uPARAP may be involved in glioma cell invasiveness through actin cytoskeletal rearrangement. downregulation of uPARAP may be a novel anti-invasion therapeutic strategy for malignant gliomas.     

Downregulation of NDRG1 promotes invasion of human gastric cancer AGS cells through MMP-2

The N-myc downstream-regulated gene-1 (NDRG1) has recently been proposed as a metastasis suppressor, but its precise role remains unclear. To investigate whether NDRG1 can indeed influence the metastasis progress, expression of endogenous NDRG1 was knocked down in human AGS gastric adenocarcinoma cells using RNA interference. Stable NDRG1 “silenced” transfectants showed similar growth rates as their control counterparts. By contrast, invasive ability in Matrigel invasion activity and Gelatinolytic activity by matrix metalloproteinase-2 (MMP-2) were markedly increased in NDRG1 “silenced” cells. Moreover, re-expression of NDRG1 by recombinant adenovirus Ad-NDRG1 in NDRG1 “silenced” cells inhibited the increased invasive ability. Further study, we found the induction of MMP-2 by downregulation of NDRG1 was mediated by MT1-MMP. Altogether, our results imply that NDRG-1 could play a key role in the regulation of cellular invasion and metastasis, which may involve the upregulation of matrix metalloproteinases.     

Reduction of Akt2 inhibits migration and invasion of glioma cells

Malignant gliomas have a tendency to invade diffusely into surrounding healthy brain tissues, thereby precluding their successful surgical removal. The serine/threonine kinase Akt2 is well known as an important regulator of cell survival and growth. In this study, we show that siRNA‐mediated depletion of Akt2 inhibited migration and invasion of glioma cells. In addition, we demonstrate the mechanisms by which Akt2 functions to promote cell migration and invasion. Phosphorylation of cofilin, a critical step of actin polymerization, and phosphorylation of Girdin, essential for the integrity of the actin cytoskeleton and cell migration, were impaired. Furthermore, epidermal growth factor‐induced ACAP1 phosphorylation and integrin β1 phosphorylation were also blocked, consistent with defects in adhesion. Thus, Akt2 regulates both cell adhesion and cytoskeleton rearrangement during migration. Decreased MMP‐9 expression in Akt2 knocked‐down glioma cells was subsequently confirmed by Western blotting, consistent with the decreased invasion in vitro and in vivo. These results suggest that Akt2 contributes to glioma cells migration and invasion by regulating the formation of cytoskeleton, influencing adhesion and increasing expression of MMP‐9. Our immunohistochemistry results by using human gliomas tissue sections also indicated that Akt2 expression was closely related with the malignancy of gliomas. This is coincident with our in vivo and in vitro results from cell lines. All of these results indicate that Akt2 is a critical factor in gliomas invasion. This study identifies that Akt2 is a potentially antiinvasion target for therapeutic intervention in gliomas. © 2009 UICC