Overexpression of the 18A2/mts1 gene and down–regulation of the TIMP–2 gene in invasive human glioma cell lines in vitro

Invasion of the reconstituted extracellular matrix composite, Matrigel, by eight human glioma–derived cell lines and human fetal brain cells was assessed in vitro using 8 um polycarbonate filters in a modified Boyden migration chamber. With the exception of one low grade glioma derived cell line, all lines studied proved to be invasive while normal fetal brain cells failed to invade. This invasive potential was independent of the histological grade of the tumour from which the cell lines originated. In addition, the expression of the metastasis–associated gene 18A2lmts1 as well as the tissue inhibitor of metalloproteinases–2 (TIMP–2) was analysed in each of the glioma–derived cell lines. The 18A2/mtsl was expressed in all the cells studied with the exception of fetal brain cells and the low grade non–invasive glioma derived IPRK–7 cell line. The 18A2/mtsl related genes coding for the S100 subfamily of calcium binding proteins were found to be differentially and overexpressed in invasive cell lines. TIMP–2 was expressed only in noninvasive cell lines. These results suggest that the 18A2/ mtsl and TIMP–2 genes could play an important role in the invasive behaviour of human glioma cells in vitro. .     

Vascular endothelial growth-factor production is stimulated in response to growth-factors in human glioma-cells

Neovascularization is essential for tumour growth and is mediated by physiological substances produced by tumours. Vascular endothelial growth factor (VEGF) is one such potent angiogenic factor. Human gliomas, the most important class of intrinsic brain tumours, express VEGF both in vivo and in vitro. Factors involved in the control of VEGF production by glioma cells are not well known. In this study, we investigated the role of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and platelet derived growth factors (PDGF) on VEGF production by four different glioma cell lines in vitro. With the exception of PDGF A/A and B/B in one cell line, all growth factors differentially stimulated VEGF production in all cell lines investigated. These data suggest that VEGF production in human glioma may be regulated by other growth factors which are also known to be expressed in such tumours.     

Hyaluronic acid modulates glioma cell proliferation through its interaction with CD44H in vitro

In a recent study we showed that hyaluronic acid (HA) induces cell detachment and promotes migration and invasion of glioma cells in vitro through its interaction with the cell surface molecule CD44H. In this study, the role of HA in proliferation in eight human glioma-derived cell Lines was investigated. We demonstrate that HA exerts a dose dependent proliferative and anti-proliferative effect on glioma cells. This effect was found to be partially counteracted by a CD44H monoclonal antibody, suggesting the involvement of its high affinity receptor, CD44H and other HA receptors in this process.     

The geldanamycins are potent inhibitors of the hepatocyte growth factor/scatter factor-met-urokinase plasminogen activator-plasmin proteolytic network

The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), have been implicated in human tumor development and metastasis. HGF/SF induces the expression of urokinase plasminogen activator (uPA) and the uPA receptor (uPAR), important mediators of cell invasion and metastasis. We have developed a cell-based assay to screen for inhibitors of this signaling system using the induction of endogenous uPA and uPAR and the subsequent conversion of plasminogen to plasmin as the biological end point. Assay validation was established using a neutralizing antiserum to HGF/SF and a uPA inhibitor (B428), as well as inhibitors of the MKK-MAPK1/2 pathway, shown previously to be important in the induction of uPA and uPAR. Using this assay, we found several classes of molecules that exhibited inhibition of HGF/SF-dependent plasmin activation. However, we discovered that certain members of the geldanamycin family of anisamycin antibiotics are potent inhibitors of HGF/SF-mediated plasmin activation, displaying inhibitory properties at femtomolar concentrations and nine orders of magnitude below their growth inhibitory concentrations. At nanomolar concentrations, the geldanamycins down-regulate Met protein expression, inhibit HGF/SF-mediated cell motility and invasion, and also revert the phenotype of both autocrine HGF/SF-Met transformed cells as well as those transformed by Met proteins with activating mutations. Thus, the geldanamycins may have important therapeutic potential for the treatment of cancers in which Met activity contributes to the invasive/metastatic phenotype.     

Establishment and characterization of a primary androgen-responsive African-American prostate cancer cell line, E006AA

BACKGROUND: Establishment of human prostate cancer cell lines is essential to advance our understanding of complex processes associated with the initiation and progression of the disease. In the present study, we report the establishment of a primary African-American prostate cancer cell line (E006AA) as well as its associated stromal cells (S006AA). METHODS: E006AA cell line was established as a spontaneously immortalized cells from a patient with a clinically localized prostate cancer. Extensive characterization of the cells was accomplished using androgen-dependent growth and sensitivity assays, Western analyses, RT-PCR/real-time PCR, cytogenetic analyses, and tumorigenicity in nude mice. RESULTS: E006AA cell line shows androgen-dependent growth, expresses PSA and the androgen receptor (AR) with 26 CAG repeats in exon 1 of AR. Cytogenetic analyses revealed a hypertriploid karyotype with additional numerical gains in chromosomes 5, 6, 8, 10, 17, 20, 21 and a marker chromosome of unknown origin as well as structural abnormalities in chromosomes 4, 5, 8, 9, 11-14, 18, and 20. This cell line is not tumorigenic in nude mice. S006AA cell line, isolated from the same tumor specimen, also expresses AR and shows the morphological characteristics of smooth muscle cells of prostatic stroma. CONCLUSIONS: These cell lines are the first available primary epithelial and stromal cells derived from an African-American patient with organ-confined prostate cancer and in conjunction with other established cell lines, could provide an in vitro model system to investigate early transforming events in prostate cancer.     

Hyaluronic acid/CD44H interaction induces cell detachment and stimulates migration and invasion of human glioma cells in vitro

The mechanisms underlying the invasive properties of glio-mas, the major form of intrinsic brain tumours in humans, are poorly understood. We have reported that CD44 plays an important role in this behaviour in vitro. In the present work, we investigated the role of its ligand, hyaluronic acid (HA), in invasion in 8 human glioma cell lines. We found that HA mediates cell detachment via its interaction with its high affinity receptor, CD44H. Using 8 μm porosity polycarbonate filter transwells, we demonstrate that HA strongly stimulates migration in all 8 cell lines. This effect was found to be partially counteracted by a CD44H monoclonal antibody (MAb), suggesting the involvement of CD44H, as well as other HA receptors, in this process. Furthermore, incorporation of increasing concentrations of HA in Matrigel in an in vitro invasion assay resulted in a substantial increase in the invasive propensity of the glioma cell lines. Moreover, blocking experiments with the CD44H MAb suggest that CD44H and other receptors interact with HA to promote cell invasion in vitro. Our results show that HA induces cell detachment, stimulates migration and promotes invasion via its interaction with CD44H and other HA receptors in vrtro. These effects could be prevented by use of specific HA receptor antibodies.     

Gangliosides modulate proliferation,migration, and invasiveness of human brain tumor cells in vitro

Gliomas, the most common form of intrinsic brain tumor, are characterized by diffuse local invasion of the normal brain structures, irrespective of their histological grade of malignancy; a feature that is a major obstacle to successful therapy. They generally infiltrate the central nervous system (CNS) as individual tumor cells several centimeters beyond the macroscopic tumor margin and consequently often recur, after subtotal surgical resection. Factors involved in the control of both their proliferation and invasiveness are poorly documented. In this work, the role of gangliosides on proliferation of both human fetal human brain cells and five cell lines derived from human gliomas with different grades of malignancy was investigated. In addition, 8 μm-porosity polycarbonate filters were used to study cell motility. In addition, these filters were coated with the reconstituted extracellular matrix (ECM) composite, Matrigel, to assess invasiveness. The results presented show that gangliosides generally exert a proliferation inhibitory effect on fetal brain cells and glioma cell lines in vitro and play an important role in promoting glioma cell motility and invasiveness. The molecular mechanisms involved in the action of gangliosides may prove useful in identifying new targets for an anti-invasion therapy.     

Prosaposin upregulates AR and PSA expression and activity in prostate cancer cells (LNCaP)

BACKGROUND: Prosaposin overexpression and/or genomic amplification have been demonstrated in androgen-independent (AI) prostate cancer cell lines and tissues. Here, we explored the possibility for a functional relationship between prosaposin and androgen receptor (AR) in LNCaP cells. METHODS: The effect of prosaposin or its active molecular derivatives (e.g., saposin C) on expression and activity of androgen receptor (AR) and prostate-specific antigen (PSA) was examined by using immunoblotting, RT-PCR, transfection, and reporter gene assays, immunofluorescence staining, and inhibitors of signal transduction pathways. RESULTS: Prosaposin or saposin C, in an AI-manner, (a) increased AR mRNA and protein expression and nuclear AR content and its phosphorylation state; (b) increased PSA mRNA and protein expression; and (c) upregulated PSA- and an androgen-inducible probasin (PB)-reporter gene activity in LNCaP and AR-transfected PC-3 cells. Induction of PSA expression and reporter activity was substantially blocked or prevented with the antiandrogen bicalutamide, pertussis toxin, or inhibitors of MAPK- and PI3K/Akt-signaling pathways, indicating an androgen-agonistic effect for saposin C that involves AR and multiple signaling pathways. CONCLUSIONS: The results for the first time introduce prosaposin as an androgen-agonist in prostate cancer cells. This finding, together with the growth-promoting effect and overexpression of prosaposin, may support a growth advantage to AI prostate cancer cells.