Biomedical subjects

Niranjan Yanamandra

Publications and source records attributed to Niranjan Yanamandra.

12 recordsLinked to original sources

PubMed2003

Expression of antisense uPAR and antisense uPA from a bicistronic adenoviral construct inhibits glioma cell invasion, tumor growth, and angiogenesis.

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in the invasiveness of gliomas and other infiltrative tumors. In glioma cell lines and tumors, high grade correlates with increased expression of uPAR and uPA. We report here the downregulation of uPAR and uPA by delivery of antisense sequences of uPAR and uPA in a single adenoviral vector, Ad-uPAR-uPA (Ad, adenovirus). The bicistronic construct (Ad-uPAR-uPA) infected glioblastoma cell line had significantly reduced levels of uPAR, uPA enzymatic activity and immunoreactivity for these proteins when compared to controls. The Ad-uPAR-uPA infected cells showed a markedly lower level of invasion in the Matrigel invasion assays, and their spheroids failed to invade the fetal rat brain aggregates in the coculture system. Intracranial injection of SNB19 cells with the Ad-uPAR-uPA antisense bicistronic construct showed inhibited invasiveness and tumorigenicity. Subcutaneous injections of bicistronic antisense constructs into established tumors (U87 MG) caused regression of those tumors. Our results support the therapeutic potential of targeting the individual components of the uPAR-uPA system by using a single adenovirus construct for the treatment of glioma and other invasive cancers.

Adenoviridae↗

PubMed2003

Promoter methylation and silencing of the tissue factor pathway inhibitor-2 (TFPI-2), a gene encoding an inhibitor of matrix metalloproteinases in human glioma cells.

We have shown previously that the tissue factor pathway inhibitor-2 (TFPI-2), a broad range proteinase inhibitor, is highly expressed in low-grade gliomas, but, minimally expressed or undetectable in glioblastomas, and that enforced expression of this gene reduces the invasive properties of brain tumor cells. Here, we examined the role of promoter methylation as a mechanism of TFPI-2 gene silencing. In SNB19 glioblastoma cells, which have no detectable TFPI-2 expression, 5-aza-2'-deoxycytidine (5aC), an inhibitor of DNA methyltransferase, induced TFPI-2 mRNA in a dose-dependent manner. Trichostatin A (TSA), the histone deacetylase (HDAC) inhibitor, by itself, was more efficient than 5aC in inducing TFPI-2 transcripts, and the 5aC+TSA combination resulted in highly synergistic reactivation of the gene, both at the transcript and protein levels. In Hs683 glioma cells, which express the TFPI-2 gene at high levels, transfection of the in vitro methylated TFPI-2 promoter constructs resulted in a drastic decrease of promoter activity compared to the unmethylated promoter. Further, the methylation-specific PCR in SNB19 and Hs683 cells showed that TFPI-2 gene repression was closely linked with methylation of the CpG islands in the promoter. Finally, the chromatin immunoprecipitation assays in SNB19 cells showed that the methylated and repressed TFPI-2 promoter was associated with the methyl-CpG binding protein 2 (MeCP2), and that gene reactivation resulted in the loss of MeCP2 from this site. These studies establish that TFPI-2 is transcriptionally silenced through promoter methylation in SNB19 cells.

Azacitidine↗

PubMed2003

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.

Adenoviridae↗

PubMed2003

Triterpenoids from Glycine max decrease invasiveness and induce caspase-mediated cell death in human SNB19 glioma cells.

In recent years there has been an increasing interest in compounds present in foods that may prevent or slow the progression of chronic illnesses, such as cardiovascular disease, osteoporosis and cancer. Saponins have been reported to have important time-dependent anti-cancer properties. We have used a highly purified and characterized saponin fraction containing the soyasapogenol B glycosides (the 'B group' saponins) from soybeans (Glycine max L.) to demonstrate a reduction in SNB 19 human glioblastoma cell invasion (45% decrease compared to untreated cells) in vitro in a Matrigel invasion assay. We have also demonstrated that triterpenoid saponin induces apopotosis and affects mictochondiral function. Dose-dependent loss of mitochondrial trans-membrane potential in SNB 19 cells occurred with treatment, along with release of cytochrome c, processing of caspase-9, and -3 and specific cleavage of poly ADP-ribose polymerase (PARP), a substrate of caspase-3. The results suggest that the saponin fraction induces apoptosis in SNB19 human glioblastoma cells by stimulating cytochrome-c release and subsequent activation of a caspase cascade. Our observations clearly demonstrate the pro-apoptotic and anti-invasive activities of the soyasapogenol B glycosides from soybeans.

Annexin A5↗

PubMed2003

Expression of matrix metalloproteinases, their inhibitors, and urokinase plasminogen activator in human meningiomas.

MMP-2, MMP-9, and uPA have been previously described as important to the invasive and metastatic potential of human tumors, including breast, lung, glioblastoma, and prostate. We examined the activity of these proteases and the levels of their inhibitors (TIMP-1 and TIMP-2) in a series of human meningioma tissue samples. Normal brain tissue did not show elevated levels of uPA, MMP-2 or MMP-9 activity. Meningiomas showed a mild, to moderate to significantly high level of uPA, MMP-2, and MMP-9. However, no increase in TIMP-1 or TIMP-2 levels was detected. Immunohistochemistry confirmed the assay findings and localized these molecules to the cell surface. The findings provide evidence for elevated levels of uPA and MMPs in meningiomas and suggest a therapeutic target for minimizing the malignant propensity of meningiomas using protease inhibitors.

Brain↗

PubMed2003

Physiological and chemical inducers of tissue factor pathway inhibitor-2 in human glioma cells.

Tissue factor pathway inhibitor-2 (TFPI-2), a serine protease inhibitor abundant in the extracellular matrix, is expressed in high amounts in low-grade, non-invasive glioma cells but in low amounts in high-grade, highly invasive glioma cells. Overexpression of TFPI-2 by highly invasive glioma cells reduces their invasiveness and thus may be useful in cancer therapy. The mechanisms underlying the transcriptional regulation of TFPI-2 are not well elucidated. We previously reported that the -312 to +1 region of TFPI-2 was critical for the minimal, inducible regulation of TFPI-2 in gliomas. This region harbors sites for several transcription factors, including SP1 (-192 to -183 and -135 to -128), AP-1 (-310 to -300, -213 to -204, and -163 to -154), NF-kappaB (-229 to -221), an NF-kappaB-like site (-291 to -281), and Lyf-1 (-260 to -252). Here we transiently transfected low-grade Hs683 glioma cells with mutant constructs to clarify the role of these transcription factors in TFPI-2 regulation. Addition of phorbol 12-myristate 13-acetate, 1,2-diacyl-sn-glycerol, IFN-gamma, or IFN-alpha induced the expression of TFPI-2 wild-type promoter construct as well as TFPI-2 protein and mRNA in Hs683 cells. Mutations at either of two AP-1 sites (-310 to -300 and -163 to -154) or either of two SP1 sites (-192 to -183 and -135 to -128) resulted in reduced TFPI-2 activity, regardless of the presence of stimulator compounds, and reduction in DNA-protein binding (by electrophoretic mobility shift assay).

Base Sequence↗

PubMed2002

Modulation of cystatin C expression impairs the invasive and tumorigenic potential of human glioblastoma cells.

Increases in the abundance of cathepsin B transcript and protein with increased tumor grade and changes in subcellular localization and activity of this enzyme. We observed progressive reductions in levels of the protease inhibitor cystatin C, an inhibitor of cathepsin B with corresponding increases in the malignancy of glioma cell lines, implying an inverse correlation between cystatin C and tumor grade. To investigate the role of cystatin C in the invasion of brain tumor cells, we stably transfected SNB19 glioblastoma cells with either a 0.4-kb cDNA construct of human cystatin C in the sense orientation or an empty vector. Clones expressing sense-cystatin C cDNA had higher cystatin C mRNA and protein levels than did control cells. Sense-transfected cells were also markedly less invasive than control cells in a Matrigel invasion assay and in a coculture assay of SNB19 spheroids and fetal rat brain aggregates. Finally, the sense-transfected cells did not form tumors in nude mice upon intracerebral injection. These results strongly implicate cystatin C in the invasiveness of human glioblastoma cells and suggest that sense transcripts of cystatin C may prove useful in cancer therapy.

Animals↗

PubMed2002

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.

Adenoviridae↗

PubMed2002

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.

Actins↗

PubMed2002

The mutational spectrum of brachydactyly type C.

Growth/differentiation factor-5 (GDF5), also known as cartilage-derived morphogenetic protein-1 (CDMP-1), is a secreted signaling molecule that participates in skeletal morphogenesis. Heterozygous mutations in GDF5, which maps to human chromosome 20, occur in individuals with autosomal dominant brachydactyly type C (BDC). Here we show that BDC is locus homogeneous by reporting a GDF5 frameshift mutation segregating with the phenotype in a family whose trait was initially thought to map to human chromosome 12. We also describe heterozygous mutations in nine additional probands/families with BDC and show nonpenetrance in a mutation carrier. Finally, we show that mutant GDF5 polypeptides containing missense mutations in their active domains do not efficiently form disulfide-linked dimers when expressed in vitro. These data support the hypothesis that BDC results from functional haploinsufficiency for GDF5.

Animals↗

PubMed2002

Minimal and inducible regulation of tissue factor pathway inhibitor-2 in human gliomas.

Tissue factor pathway inhibitor-2 (TFPI-2), a serine protease inhibitor abundant in the extra cellular matrix, is highly expressed in non-invasive cells but undetectable levels in highly invasive human glioma cells. The mechanisms responsible for its transcriptional regulation are not well elucidated. In this study, we made several deletion constructs from a 3.6 kb genomic fragment from Hs683 cells containing the 5'-flanking region of the TFPI-2 gene, transiently transfected with these constructs into non-invasive (Hs683) and highly invasive (SNB19) human glioma cells, and assessed their expression by using a luciferase reporter gene. Three constructs showed high promoter activity (pTF5, -670 to +1; pTF6, -312 to +1; pTF2, -1511 to +1). Another construct, pTF8 (-81 to +1), showed no activity. PTF9, a variant of pTF5 in which a further 231 bp fragment (-312 to -81) was deleted, from the [-670 to +1] pTF5 region, also showed no promoter activity. Hence, (-312 to -81) this region is essential for the transcription of TFPI-2 in glioma cells. Sequencing of this promoter region revealed that it has a high G+C content, contains potential SP1 and AP1 binding motifs, and lacks canonical TATA and CAAT boxes immediately upstream of the major transcriptional initiation site, although CAAT boxes were found about -3000 bp upstream of the transcription start site. We also found a strong repressor in the region between -927 to -1181, upstream of the major transcriptional initiation site, followed by positive elements or enhancers between -1511 to -1181. These positive elements masked the silencer effect. Finally TFPI-2 was induced in Hs683 cells transfected with the pTF6 construct (-312 to +1) and stimulated with phorbol-12-myristate-13-acetate (PMA). We conclude that the -312 to +1 region is critical for the minimal and inducible regulation of TFPI-2 in non-invasive (Hs683) and highly invasive (SNB19) human glioma cell lines.

Base Sequence↗

PubMed2002

Suppression of glioma invasion and growth by adenovirus-mediated delivery of a bicistronic construct containing antisense uPAR and sense p16 gene sequences.

Our previous studies showed that the urokinase-type plasminogen activator receptor (uPAR) and the p16 tumor suppressor gene play a significant role in glioma invasion. We expected that downregulation of uPAR and overexpression of p16 using a bicistronic vector might cause a additive and cooperative effect in the suppression of glioma invasion and growth. The bicistronic construct (Ad-uPAR/p16)-infected glioblastoma cell lines had significantly lower levels of uPAR and higher levels of p16 than controls. Cell cycle analysis showed the bicistronic vector caused G0/G1 arrest of the cell cycle. In vitro glioblastoma cell growth and invasiveness were inhibited in Ad-uPAR/p16-infected cells compared with controls. Ad-uPAR/p16 suppressed the tumor growth of glioblastoma cell lines in an ex vivo intracerebral tumor model and an in vivo subcutaneous tumor model. Our results support the therapeutic potential of simultaneously targeting uPAR and p16 in the treatment of gliomas.

Adenoviridae↗