Blockade of cathepsin B expression in human glioblastoma cells is associated with suppression of angiogenesis.

The cysteine proteinase cathepsin B has been implicated in tumor progression by virtue of its increased mRNA and protein levels, as well as its localization at the invading front of the tumor. In this study, we examined whether blocking cathepsin B expression in human glioblastoma SNB19 cells affects angiogenesis. Stable transfectants of human glioblastoma cells with a plasmid containing antisense cathepsin B cDNA showed decreased migration rates in wound- and spheroid-migration assays. Analysis showed a reduction in VEGF protein and MMP-9 activity in the cathepsin B antisense cDNA-transfected cells. Regarding angiogenesis in vitro, we found that the conditioned medium of glioblastoma cells with downregulated cathepsin B expression reduced cell-cell interaction of human microvascular endothelial cells, resulting in the disruption of capillary-like network formation. Furthermore, a marked reduction in microvasculature development was seen in an in vivo dorsal air sac assay of glioblastoma cells with downregulated cathepsin B expression. Taken together, these results provide evidence that inhibition of cathepsin B expression can suppress glioblastoma-induced neovascularization.     

Modulation of invasive properties of murine squamous carcinoma cells by heterologous expression of cathepsin B and cystatin C.

Murine SCC-VII squamous carcinoma cells have the capacity to penetrate reconstituted basement membranes (Matrigel) in vitro. The invasion of Matrigel layers by SCC-VII cells was significantly reduced by E-64, a specific inhibitor of lysosomal cysteine proteinases. The cathepsin-B-selective E-64 derivative, CA-074, inhibited penetration of Matrigel by SCC-VII cells to the same extent, indicating a major role for this particular lysosomal enzyme in extracellular-matrix degradation during squamous-carcinoma-cell invasion. SCC-VII cells were stably transfected with a cDNA encoding human procathepsin B, in an attempt to modulate the invasive properties of the cell line. The transfected cells expressed the heterologous gene, secreted increased amounts of procathepsin B and displayed enhanced invasive potential. In vivo, the activity of cathepsin B is strictly regulated by endogenous inhibitors. SCC-VII cells were therefore also stably transfected with a cDNA encoding human cystatin C, the most potent cysteine-proteinase inhibitor in mammalian tissues. The expression of this transgene resulted in the production of active recombinant cystatin C and a pronounced reduction in Matrigel invasion. These studies demonstrate that the invasive properties of squamous-cell carcinomas can be changed by modulation of the balance between cathepsin B and its endogenous inhibitors, and provide further evidence for the involvement of this lysosomal cysteine proteinase in tumour invasion and metastasis.     

Heparanase affects adhesive and tumorigenic potential of human glioma cells

Heparanase is an endo-beta-glucuronidase responsible for the cleavage of heparan sulfate, participating in extracellular matrix degradation and remodeling. Traditionally, heparanase activity was well correlated with the metastatic potential of a large number of tumor-derived cell types. More recently, heparanase up-regulation was detected in essentially all human tumors examined, correlating, in some cases, with poor postoperative survival and increased tumor vascularity. The role of heparanase in primary tumor progression is, however, poorly understood. Here, we overexpressed the human heparanase gene in a human glioma cell line, U87. We found that heparanase overexpression induces cell invasion, as might be expected. Surprisingly, elevated heparanase expression levels correlated with decreased proliferation rates and increased cell spreading and formation of a tight monolayer rather than large cell aggregates. This phenotypic appearance was accompanied by beta1-integrin activation, FAK and Akt phosphorylation, and Rac activation. In a xenograft tumor model, relatively moderate heparanase expression levels significantly enhanced tumor development and tumor vascularity, whereas high heparanase expression levels inhibited tumor growth. These results indicate that heparanase activates signal transduction pathways and, depending on its expression levels, may modulate tumor progression.     

Down-regulation of uPAR and cathepsin B retards cofilin dephosphorylation

Cathepsin B and uPAR play key roles in cancer cell migration and invasion. Here, we demonstrate that the simultaneous, siRNA-mediated down-regulation of uPAR and cathepsin B inhibits glioma cell migration and is accompanied by cytoskeletal condensation. We show that the dephosphorylation of cofilin is inhibited by the down-regulation of uPAR alone and, to a lesser extent, by the down-regulation of cathepsin B alone, and that the effect was much higher with the down-regulation of both molecules by pUC. Using FACS analysis and western blotting for the alphaVbeta3 integrin heterodimer, we determined that down-regulating uPAR subsequently causes the down-regulation of the alphaVbeta3 integrin heterodimer. As evidenced by western blot analysis of ERK1/2, pERK1/2, p38MAPK, p-p38MAPK, AKT, pAKT and PI3-k, the MEK and PI3-k pathways are inhibited. From cytoskeleton studies, we observed that the down-regulation of uPAR caused cytoskeletal condensation and that the simultaneous down-regulation of uPAR and cathepsin B was even more effective at inducing cytoskeletal condensation than uPAR alone. Our results demonstrate the relevance of uPAR in cytoskeletal dynamics and the potential of uPAR and cathepsin B as targets in the treatment of malignant gliomas.     

Inhibitory effects of cyclosporin A on calcium mobilization-dependent interleukin-8 expression and invasive potential of human glioblastoma U251MG cells

Interleukin (IL)-8 produced from glioblastoma is suggested to contribute to its own proliferation and progression. Since various external stimuli have been shown to increase intracellular Ca(2+) in glioma cells, we investigated Ca(2+) mobilization-dependent IL-8 expression and effect of cyclosporin A (CsA), an inhibitor of calcineurin (Cn), on the expression and invasive potential of human glioblastoma U251MG cells. Combined treatment with Ca(2+)-ionophore and phorbol-myristate-acetate (A23187/PMA) increased IL-8 mRNA and protein levels. This increase was suppressed by CsA and by another Cn inhibitor FK506. Luciferase reporter gene assay and electrophoretic mobility shift assay revealed that activation of p65-containing nuclear factor-kappaB was essential for A23187/PMA-dependent activation of IL-8 promoter. CsA suppressed the promoter activity by attenuating IkappaB-alpha degradation. U251MG cells expressed IL-8 receptors CXCR-1 and -2, and Matrigel invasion assay revealed that CsA attenuated A23187/PMA-dependent stimulation of invasive potential, probably by inhibiting IL-8 production. In addition, IL-8-dependent proliferation was also suppressed by CsA. Taken together, these results demonstrate the novel inhibitory effects of CsA on glioblastoma cell functions, suggesting CsA as a potential therapeutic adjuvant for glioma treatment.     

A comparison of the expression of the DIDS-binding proteins from normal and tumorigenic human cells.

A monoclonal antibody was prepared against DIDS, an inhibitor of anion transport, and used to compare the occurrence and distribution of DIDS-binding sites of tumorigenic and non-tumorigenic human somatic-cell hybrids. The monoclonal antibody (E8) was produced by the fusion of the mouse myeloma (NS-1) with mouse spleen cells and is of the IgG1 subclass. The apparent half-saturation of DIDS for HEp-2 cells is 16 microM and the reaction is rapid. The number of binding sites on tumorigenic and non-tumorigenic hybrid cells was the same. The DIDS-binding protein occurs homogeneously on all cells, a characteristic which distinguishes it from the possible tumour antigen recognised by the M/27 monoclonal antibody.     

Ribozyme-mediated inhibition of survivin expression increases spontaneous and drug-induced apoptosis and decreases the tumorigenic potential of human prostate cancer cells

Survivin is a member of the inhibitor of apoptosis protein (IAP) family, which has been implicated in inhibition of apoptosis and control of mitotic progression. The finding that survivin is overexpressed in most human tumors but absent in normal adult tissues has led to the proposal of survivin as a promising therapeutic target for anticancer therapies. We decided to evaluate the effects of a ribozyme-based strategy for survivin inhibition in androgen-independent human prostate cancer cells. We constructed a Moloney-based retroviral vector expressing a ribozyme targeting the 3' end of the CUA(110) triplet in survivin mRNA, encoded as a chimeric RNA within adenoviral VA1 RNA. Polyclonal cell populations obtained by infection with the retroviral vector of two androgen-independent human prostate cancer cell lines (DU145 and PC-3) were selected for the study. Ribozyme-expressing prostate cancer cells were characterized by a significant reduction of survivin expression compared to parental cells transduced with a control ribozyme; the cells became polyploid, underwent caspase-9-dependent apoptosis and showed an altered pattern of gene expression, as detected by oligonucleotide array analysis. Survivin inhibition also increased the susceptibility of prostate cancer cells to cisplatin-induced apoptosis and prevented tumor formation when cells were xenografted in athymic nude mice. These findings suggest that manipulation of the antiapoptotic survivin pathway may provide a novel approach for the treatment of androgen-independent prostate cancer.     

V3 versican isoform expression alters the phenotype of melanoma cells and their tumorigenic potential

Versican is a large chondroitin sulfate proteoglycan produced by several tumor cell types, including malignant melanoma. The expression of increased amounts of versican in the extracellular matrix may play a role in tumor cell growth, adhesion and migration. We have expressed the V3 isoform of versican in human and canine melanoma cell lines. Retroviral overexpression of V3 did not change the morphology of any of the cell lines but markedly reduces cell growth in the V3 versican expressing melanoma cells. The V3-overexpressing melanoma cells retain their diminished growth potential in vivo because primary tumors arising from these cell lines growth more slowly than their vector only counterparts. This effect was accompanied by increases in cell adhesion on hyaluronan and an enhanced ability to migrate on hyaluronan-coated transwell chambers. This enhanced migration is blocked when cells are preincubated with soluble hyaluronan, or anti-CD44 antibodies, suggesting that V3 acts by altering the hyaluronan-CD44 interaction. Hyaluronan content and CD44 expression are not altered in V3-overexpressing cells compared to vector-transduced cells. Our results show that V3 overproduction modulates the in vitro behavior of human and canine melanoma cell lines and reduces their tumorigenicity in vivo.     

Alterations of p53 in tumorigenic human bronchial epithelial cells correlate with metastatic potential.

The cellular and molecular mechanisms of radiation-induced lung cancer are not known. In the present study, alterations of p53 in tumorigenic human papillomavirus-immortalized human bronchial epithelial (BEP2D) cells induced by a single low dose of either alpha-particles or 1 GeV/nucleon (56)Fe were analyzed by PCR-single-stranded conformation polymorphism (SSCP) coupled with sequencing analysis and immunoprecipitation assay. A total of nine primary and four secondary tumor cell lines, three of which were metastatic, together with the parental BEP2D and primary human bronchial epithelial (NHBE) cells were studied. The immunoprecipitation assay showed overexpression of mutant p53 proteins in all the tumor lines but not in NHBE and BEP2D cells. PCR-SSCP and sequencing analysis found band shifts and gene mutations in all four of the secondary tumors. A G-->T transversion in codon 139 in exon 5 that replaced Lys with Asn was detected in two tumor lines. One mutation each, involving a G-->T transversion in codon 215 in exon 6 (Ser-->lle) and a G-->A transition in codon 373 in exon 8 (Arg-->His), was identified in the remaining two secondary tumors. These results suggest that p53 alterations correlate with tumorigenesis in the BEP2D cell model and that mutations in the p53 gene may be indicative of metastatic potential.     

Interactions between human colon carcinoma cells, fibroblasts and monocytic cells in coculture--regulation of cathepsin B expression and invasiveness

To elucidate the importance of the tumor/host interaction in malignant tumors, we investigated the colon carcinoma cell line HT-29 in coculture with monocytic cells (THP-1) and fibroblasts (175BR) for cathepsin B expression and activity. The tumor cells were grown in monolayer cultures or as multicellular tumor spheroids. After coculture, the three cell types were separated by labeled magnetic beads for cathepsin B mRNA and protein analysis. The invasive potential was studied in in vitro invasion assays. The expression level of cathepsin B was found to be 10-fold increased in three dimensional spheroids of HT-29 compared to HT-29 monolayers. The coculture of HT-29 with THP-1 cells and/or human fibroblasts led to a considerable increase in cathepsin B mRNA expression in both tumor and tumor-associated cells. The invasive potential of the tumor cells was 5 times increased by adding monocytic cells to the assay system. This is dependent on the functional activity of cathepsin B as shown by specific siRNA's and seems to be regulated by activation of ERK1/2 and p38 signal transduction pathways.     

Irradiated mammary gland stroma promotes the expression of tumorigenic potential by unirradiated epithelial cells

We have shown that ionizing radiation, a known carcinogen of human breast, elicits rapid, persistent, and global changes in the mammary microenvironment as evidenced by altered extracellular matrix composition and growth factor activities. To address whether these events contribute to radiogenic carcinogenesis, we evaluated the effect of irradiated mammary stroma on the neoplastic potential of COMMA-D mammary epithelial cells. Although COMMA-D cells harbor mutations in both alleles of p53, they are nontumorigenic when injected s.c. into syngeneic hosts. Unirradiated COMMA-D cells transplanted to mammary fat pads cleared previously of epithelia preferentially formed tumors in irradiated hosts. Tumor incidence at 6 weeks was 81% +/- 12 SE when animals were irradiated with 4 Gy, 3 days prior to transplantation, compared with 19% +/- 2 SE (P < 0.005) in sham-irradiated hosts. This effect was evident when cells were transplanted 1 to 14 days after irradiation. Furthermore, tumors were significantly larger (243.1 +/- 61.3 mm3 versus 30.8 +/- 8.7 mm3) and arose more quickly (100% by 6 weeks versus 39% over 10 weeks in sham hosts) in fat pads in irradiated hosts. The contribution of local versus systemic effects was evaluated using hemibody (left versus right) irradiation; tumors formed only in fat pads on the irradiated side. These data indicate that radiation-induced changes in the stromal microenvironment can contribute to neoplastic progression in vivo. Disruption of solid tissue interactions is a heretofore unrecognized activity of ionizing radiation as a carcinogen.     

Correlation of the invasive potential of glioblastoma and expression of caveola-forming proteins caveolin-1 and CAVIN1

Journal of Neuro-Oncology - Glioblastoma (GBM) is the most common primary brain cancer. The average survival time for the majority of patients is approximately 15&nbsp;months after diagnosis. A...     

Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma

Transformed stem cells have been isolated from some human cancers. We report that, unlike other brain cancers, the lethal glioblastoma multiforme contains neural precursors endowed with all of the critical features expected from neural stem cells. Similar, yet not identical, to their normal neural stem cell counterpart, these precursors emerge as unipotent (astroglial) in vivo and multipotent (neuronal-astroglial-oligodendroglial) in culture. More importantly, these cells can act as tumor-founding cells down to the clonal level and can establish tumors that closely resemble the main histologic, cytologic, and architectural features of the human disease, even when challenged through serial transplantation. Thus, cells possessing all of the characteristics expected from tumor neural stem cells seem to be involved in the growth and recurrence of adult human glioblastomas multiforme.     

NF-kappaB activity blockade impairs the angiogenic potential of human pancreatic cancer cells

The effect of blockade of NF-kappaB activity on human pancreatic cancer angiogenesis was determined in an orthotopic xenograft model. Highly metastatic L3.3 human pancreatic cancer cells, which expressed an elevated level of constitutive NF-kappaB activity, were transfected with a mutated IkappaBalpha (IkappaBalphaM). After implantation in the pancreas of nude mice, parental (L3.3) and control vector-transfected (L3.3-Neo) cells produced rapidly growing tumors and liver metastases, whereas IkappaBalphaM-transfected (L3.3-IkappaBalphaM) cells had decreased tumorigenicity and metastatic potential. NF-kappaB signaling blockade significantly inhibited the in vitro and in vivo expression of the major proangiogenic molecules vascular endothelial growth factor and interleukin-8 and decreased tumor vascular formation. These events were correlated with retarded tumor growth and suppression of metastasis. Collectively, these data suggest that suppression of tumorigenicity and metastasis by NF-kappaB blockade is due to impaired angiogenic potential of tumor cells.