Nature of the interaction of growth factors with suramin.

Suramin inhibits the binding of a variety of growth factors to their cell surface receptors. The direct interaction of suramin with acidic fibroblast growth factor has been detected by the enhancement of the drug's fluorescence in the presence of the protein with the maximum effect occurring at a molar ratio of suramin to aFGF of 2:1. This interaction stabilizes aFGF to thermal denaturation and partially protects a free thiol in its polyanion binding site from oxidation. The binding of suramin to aFGF also induces aggregation of the growth factor to at least a hexameric state as detected by static and dynamic light scattering as well as by gel filtration studies. Both CD and amide I' FTIR spectra of aFGF in the presence and absence of suramin suggest that the drug may also be causing a small conformational change in the growth factor. Suramin produces an even greater aggregation of bFGF and PDGF but not of EGF or IGF-1. Evidence for a suramin-induced conformational change in IGF-1 but not EGF is found by CD, however. It is concluded that suramin binds to many growth factors and that this induces microaggregation and, in some cases, conformational changes. In the case of aFGF, suramin interacts at or near its heparin binding site. The relationship between these phenomena and the anti-growth factor activity of suramin remains to be clearly elucidated.     

Suramin inhibition of growth factor receptor binding and mitogenicity in AKR-2B cells

Suramin, a polyanionic compound, has previously been shown to dissociate platelet-derived growth factor (PDGF) from its receptor. In the present study suramin was found to inhibit the growth of sparse cultures of AKR-2B cells in fetal bovine serum (FBS)-supplemented medium in a dose-dependent, reversible fashion. Suramin also inhibited the ability of FBS, transforming growth factor beta (TGF beta), heparin-binding growth factor type-2 (HBGF-2), and epidermal growth factor (EGF) to stimulate DNA synthesis in density-arrested cultures of AKR-2B cells. The inhibition of growth factor-stimulated mitogenicity was directly correlated to the dose of suramin required to inhibit the binding of 125I-labeled TGF beta, HBGF-2, and EGF to their cell surface receptors. Suramin affected TGF beta and HBGF-2-related events at a 10-15-fold lower dose than that required for EGF-related events. It was also noted that suramin inhibited TGF beta-stimulated soft agar colony formation of AKR-2B (clone 84A) cells as well as the spontaneous colony formation of AKR-MCA cells, a chemically transformed derivative of AKR-2B cells. This demonstrates that suramin's spectrum of action for growth factors and their receptors should be extended to include TGF beta, HBGF-2, and EGF as well as PDGF. The data further suggest that the spontaneous growth of AKR-MCA cells in soft agar is dependent on growth factor binding to cell surface receptors.     

Effect of suramin on growth of androgen-responsive mouse tumor (Shionogi carcinoma 115) and its autonomous subline (Chiba subline 2)

Suramin has been shown to inhibit the binding of various growth factors to their receptors. Shionogi Carcinoma 115 cells (SC 115 cells) and Chiba Subline 2 cells (CS 2 cells) are clones of an androgen-responsive mouse tumor cell and its autonomous subline, respectively. Since the growth of SC 115 and CS 2 cells are assumed to be regulated by their own fibroblast growth factor (FGF)-like growth factors, the present study was undertaken to examine the effect of suramin on these cells. Suramin inhibited the growth of SC 115 and CS 2 cells in a dose dependent manner. The inhibition of suramin was reversible up to 50 micrograms/ml. Suramin reversibly changed the shape of these cells from fibroblast-like to polygonal and epithelial-like ones, and inhibited 3H-thymidine incorporation into these cells which was evoked by acidic and basic FGFs, and conditioned medium obtained from CS 2 cells. The binding of 125I-basic FGF to SC 115 and CS 2 cells was inhibited by suramin. However, suramin had no effect on growth factor production and the hst-1 gene expression on CS 2 cells. In conclusion, suramin inhibited the autocrine and paracrine growth of SC 115 and CS 2 cells by blocking the binding of autocrine growth factors to their receptors.     

The effect of azidothymidine on germ tube formation in Candida albicans

Candida albicans have a marked propensity to cause infections in AIDS patients. A virulent trait of C. albicans is the yeast-hypha transition (Y-->H) which is influenced, in vitro and in vivo, by several factors. Since azidothymidine (AZT) is used in HIV-positive patients, the effect, in vitro, of different concentrations of AZT on C. albicans Y-->H transition was evaluated. C. albicans isolated from HIV-negative and HIV-positive patients were used and strains of C. tropicalis isolated from HIV-positive patients were also tested. AZT concentrations from 0.01 microg/ml to 10 microg/ml did not have any influence on the Y-->H transition, whereas 100 microg/ml AZT significantly inhibited the germ tube formation. AZT did not influence the formation of pseudohyphae in C. tropicalis. It is suggested that C. albicans infection observed in HIV-positive patients was not influenced by AZT therapy, because at currently used dosages, the Y-->H transition was not expected to increase.     

Candida albicans and HIV-1 Infection

Candida albicans virulence is in part mediated by fibronectin (FN) interaction. We compared the adherence level to FN (using Becton Dickinson FN-coated plates) of several strains of yeast isolated from HIV-1 infected or uninfected subjects affected by candidiasis (30 strains from HIV+ subjects and 18 from HIV- subjects). More adhesive strains were found in HIV+ patients than in HIV- subjects. In particular a mean increase of 120 per cent as regards the total number of adhesive cells and 230 per cent as regards the adhesive cells producing germ tubes was detected in the former group of strains as compared to the latter ( p < 0.001 in both cases). The enhancement of FN expression induced by HIV-1 infection, as we have previously demonstrated, can increase interest in the adherence to FN of C. albicans strains isolated from AIDS-affected patients. Moreover, we also underline the important role played by HIV Nef protein in increasing the C. albicans aggressiveness. In fact a significant inhibitory effect of Nef on the phagocytosis of this yeast by macrophages has been demonstrated and the oxidative processes of these cells seem to be down-regulated by this protein.     

Neural retina of chick embryo in organ culture: effects of blockade of growth factors by suramin

The neural retina is a highly organized organ whose final histoarchitecture depends on the presence of diverse growth factors and on their interactions with extracellular matrix components. However, the role of growth factors on retinal development is not fully understood. Suramin has been shown to produce diverse cellular effects via the simultaneous block of the action of several growth factors. We have therefore studied the effects of suramin on organotypic culture of chick embryo neural retina in order to gain further insights into the participation of growth factors in neural retinal development. Neural retina was incubated for 24 h with suramin at 50-200 microM and then processed to determine cell proliferation, nuclear morphology, and actin distribution. Suramin provoked extensive morphological changes revealed by a decrease in BrdU incorporation, alterations in cellular organization, and disruption of the outer limiting membrane, with the emergence of cellular elements through it. All of these effects were dose-dependent and markedly attenuated by the simultaneous presence of suramin and fibroblast growth factor 2 (FGF-2) in the culture medium. These findings indicate that suramin induces pleiotropic effects on the histoarchitecture of the chicken neural retina in organ culture and suggest that FGF-2 is one of the biological modulators involved in the maintenance of the structural organization of the chicken neural retina.     

Suramin interrupts androgen-inducible autocrine loop involving heparin binding growth factor in mouse mammary cancer (Shionogi carcinoma 115) cells

The androgen-dependent clonal cell line SC-3, derived from Shionogi carcinoma 115, secretes a fibroblast growth factor (FGF)-autocrine growth factor in response to androgen, which is able to bind to FGF receptors. In SC-3 cells, FGF receptor expression is upregulated by the SC-3-derived growth factor, providing a means of amplifying an autocrine loop of cell growth. In the present investigations, the effect of the polysulfonated naphthylurea suramin on this autocrine loop and its amplification in SC-3 cells were studied. Suramin inhibited androgen-dependent growth of SC-3 cells in a concentration-dependent fashion: ～50% inhibition was observed at 25 μM. [³H]Thymidine incorporation into the cells stimulated with partially purified SC-3-derived growth factor was inhibited by suramin in a similar way. Additionally, suramin inhibited acidic (a) or basic (b) FGF-induced cell proliferation, though relatively high concentrations were necessary to achieve the maximal inhibition. Pretreatment of SC-3 cells with suramin decreased cell surface ¹²⁵I-bFGF binding without altering dissociation constant (Kd) of the binding sites. When the cells were incubated with 250 μM suramin for 24 h, the maximum binding (Bmax) decreased to almost 50% of the control. Treatment with suramin also decreased the levels of FGF receptor-1 mRNA to a similar extent, whereas it appeared not to affect the levels of β-actin mRNA. Moreover, suramin completely blocked androgen- or bFGF-induced accumulation of FGF receptor-1 mRNA. The inhibitory effects of suramin on FGF receptor expression were reversed by simultaneous addition of high concentrations of bFGF. These results indicate that suramin exerts its potent antiproliferative action on SC-3 cells through inhibition of an androgen-inducible autocrine loop involving SC-3-derived growth factor and FGF receptor. © 1993 Wiley-Liss, Inc.     

Suramin sensitizing cells to ionizing radiation by inactivating DNA-dependent protein kinase

Here we report that suramin sensitizes LM217, MDA-MB-468, T98G and A431 cells to ionizing radiation. Suramin sensitized cells to X radiation in a dose-dependent fashion, and longer exposure to suramin before X irradiation resulted in more efficient sensitization. The dose-modifying factors calculated from the survival curves were 1.18 in LM217 cells and 1.37 in MDA-MB-468 cells. Suramin did not sensitize Scid cells that had no DNA-dependent protein kinase activity. Suramin inhibited DNA-dependent protein kinase activity in vitro and in vivo. The concentration of suramin resulting in 50% inhibition in vitro was 1.7 microM in LM217 cells and 2.4 microM in MDA-MB-468 cells. Exposure of LM217 and MDA-MB-468 cells to suramin did not affect the level of Ku70 (G22P1) or Ku80 (XRCC5), but it increased the level of DNA-PKcs(PRKDC). Suramin did not sensitize LM217 or MDA-MB-468 cells to UV radiation. Suramin's effects were not caused by accumulation of cells in a specific phase of the cell cycle. These results suggest that suramin sensitizes cells to ionizing radiation by inhibiting DNA-dependent protein kinase activity.     

Effects of suramin, an anti-human immunodeficiency virus reverse transcriptase agent, on protein kinase C. Differential activation and inhibition of protein kinase C isozymes.

Suramin inhibited protein kinase C (PKC) type I-III activity in a concentration-dependent manner. Similar inhibitory effects were observed with M-kinase, the constitutively active catalytic fragment of PKC, and autophosphorylation of PKC types I-III. Kinetic experiments indicated that suramin competitively inhibits activity with respect to ATP (Ki = 17, 27, and 31 microM, respectively) and that it can also inhibit by interaction with the substrate histone III-S. With protamine as the Pi acceptor, suramin inhibition was dependent on lipid, being approximately 4-fold less sensitive to inhibition in the absence of phosphatidylserine and diacylglycerol than in their presence. Suramin at low concentrations (10-40 microM), in the presence of Ca2+ and absence of lipid, was able to stimulate kinase activity (approximately 200-400%) in a type-dependent manner and at higher concentrations inhibited activity with histone III-S as substrate. These results indicate that suramin, a hexa-anionic hydrophobic compound, can act as a negatively charged phospholipid analog in activating PKC in the presence of Ca2+ and absence of lipid and can inhibit Ca2+/phosphatidylserine/diacylglycerol-stimulated kinase activity at higher concentrations by competing with ATP or by interaction with the exogenous substrate. Suramin inhibited cAMP-dependent protein kinase much less potently (IC50 = 656 microM) than PKC. The ability of suramin to inhibit PKC-mediated processes in intact cells was tested using the phorbol ester-stimulated respiratory burst of neutrophils as a model system. The respiratory burst of human neutrophils, when preincubated with suramin and then stimulated with phorbol ester, was inhibited in a concentration-dependent manner, suggesting that suramin may also be able to inhibit PKC-mediated processes in intact cells.     

The growth factor inhibitor suramin reduces apoptosis and cell aggregation in protein-free CHO cell batch cultures

We have previously shown that Chinese hamster ovary (CHO) cells capable of growing in medium free of exogenous proteins die by apoptosis during all stages of a batch culture (Zanghi et al., 1999). On the basis of the hypothesis that extracellular death factors might be important in apoptosis under these conditions, we examined the effect of the growth factor inhibitor and antitumor agent suramin on CHO cell growth and apoptosis in serum-free culture. Suramin protected against apoptosis during exponential growth, as indicated by the absence of DNA laddering and an increase in cell viability from roughly 70% to above 95%. Suramin also effectively dispersed cell aggregates so that single-cell suspension culture was possible. However, suramin did not protect against apoptosis during the death phase, in contrast to serum, suggesting that antiapoptotic factors in the serum remain to be discovered. The increased viable cell yield following suramin supplementation resulted in a 40% increase in product yield, based on results with cells expressing recombinant secreted alkaline phosphatase. Polysulfated compounds dextran sulfate and polyvinyl sulfate worked nearly as well as suramin in dispersing cell clumps and increasing viable cell yield, which implies that suramin's high sulfate group density may be responsible for its effects in cell culture. In addition, suramin was beneficial for long-term adaptation of CHO cells to protein-free media suspension culture, and the compound was synergistic with insulin in accelerating this adaptation time.     

Suramin changes the fate of Spemann's organizer and prevents neural induction in Xenopus laevis.

Suramin, a polyanionic compound, which has previously shown to dissociate platelet derived growth factor (PDGF) from its receptor, prevents the differentiation of neural (brain) structures of recombinants of dorsal blastopore lip (Spemann's organizer) and competent neuroectoderm. Furthermore, the suramin treatment changes the prospective differentiation pattern of isolated blastopore lip. While untreated dorsal blastopore lip will differentiate into dorsal mesodermal structures (notochord and somites), suramin treated dorsal blastopore lip will form ventral mesoderm structures, especially heart structures. The results are discussed in the context of the current opinion about the mode of action of different growth factor superfamilies.     

Antimicrobial and Biological Effects of Bomphos and Phomphos on Bacterial and Yeast Cells

In this study, the antimicrobial effects of monophosphazenes such as SM, BOMPHOS, and PHOMPHOS were examined on bacterial and yeast strains. In addition, the biological effects of these compounds were tested on the Saccharomyces cerevisiae and Candida albicans cells. The SM has an antimicrobial effect on the bacterial and yeast strains within the range of 100 and 1500 μg. When the concentration was increased, the inhibition zone expanded on the growth media (P < 0.01; P < 0.001). Like SM, BOMPHOS molecule has antimicrobial activity on the bacterial and yeast cells. The most effective concentrations of BOMPHOS on the microorganisms were observed by 1500 μg (P < 0.001). The PHOMPHOS did not effect on the bacterial and yeast cells between 100 and 1000 range, but it has an antimicrobial effect in 1500 μg. In vitro media, the biological effects of these molecules were compared with vitamin E, melatonin, and fish oil on the yeast cells. In S. cerevisiae growth media, the cell densities were increased SM, BOMPHOS, and PHOMPHOS after 20, 30, and 45 h. The highest increase in the cell density were observed in media of BOMPHOS. In C. albicans growth media, the cell density was increased by melatonin after 20, 30, and 45 h, but were decreased by other supplemental groups. Lipid level of S. cerevisiae was reduced by administered 300 and 1000 μg vitamin E and fish oil (P < 0.01). In addition, the lipid level of the same yeast cell were diminished by the 1000 μg melatonin and 300 μg PHOMPHOS (P < 0.05, P < 0.01). The lipid level of C. albicans were increased by vitamin E and BOMPHOS and fish oil, but was decreased with PHOMPHOS (P < 0.01). In conclusion, while high concentration of PHOMPHOS has antimicrobial effects on the bacterial and yeast cells, the SM and BOMPHOS have antimicrobial effects in all the concentrations. PHOMPHOS decreased the lipid level of C. albicans, but BOMPHOS increased in the the same yeast cell. In addition, the antioxidants such as vitamin E, melatonin, and fish oils have affected on the lipid synthesis of yeast cells. Copyright 2000 Academic Press.     

Suramin suppresses growth,alkaline-phosphatase and telomerase activity of human osteosarcoma cells in vitro

Neoadjuvant chemotherapy in osteosarcoma improves the survival dramatically, but there is currents drug resistance in about 25% of patients, leading researchers to investigate alternative therapy forms. Suramin has in the last two decades been used as salvage therapy in some cancers. This study was undertaken to investigate suramin as a possible salvage therapy in osteosarcoma. The effect of suramin on three human osteosarcoma cell lines (MG-63, HOS and SaOS-2) and three primary osteosarcoma cell lines isolated from biopsies was investigated. Suramin significantly inhibited cell proliferation, determined by 3H-thymidine incorporation, of osteosarcoma cells at a dose ranging from 250 to 500 microg/ml. Suramin decreased the secretion of alkaline-phosphatase after stimulation by 1,25-dihydroxy-Vitamin D(3) up to 50% and decreased telomerase activity by up to 40%. The data demonstrate that suramin has marked in vitro effects on human osteosarcoma cells supporting further clinical investigation.     

Generation of conditional lethal Candida albicans mutants by inducible deletion of essential genes

The yeast Candida albicans is the most important fungal pathogen of humans and a model organism for studying fungal virulence. Sequencing of the C. albicans genome will soon be completed, allowing systematic approaches to analyse gene function. However, techniques to define and characterize essential genes in this permanently diploid yeast are limited. We have developed an efficient method to create conditional lethal C. albicans null mutants by inducible, FLP-mediated gene deletion. Both wild-type alleles of the CDC42 or the BEM1 gene were deleted in strains that carried an additional copy of the respective gene that could be excised from the genome by the site-specific recombinase FLP. Expression of a C. albicans-adapted FLP gene under the control of an inducible promoter generated cell populations consisting of > or = 99.9% null mutants. Upon plating, these cells were unable to form colonies, demonstrating that CDC42 and BEM1 are essential genes in C. albicans. The cdc42 null mutants failed to produce buds and hyphae and grew as large, round cells instead, suggesting that they lacked the ability to produce polarized cell growth. However, the cells still responded to hyphal inducing signals by aggregating and expressing hypha-specific genes, behaviours typical of the mycelial growth form of C. albicans. Budding cells and germ tubes of bem1 null mutants exhibited morphological abnormalities, demonstrating that BEM1 is essential for normal growth of both yeast and hyphae. Inducible, FLP-mediated gene deletion provides a powerful approach to generate conditional lethal C. albicans mutants and allows the functional analysis of essential genes.     

Facilitation of the structural and functional disorders of liver lysosomes in toxic hepatitis due to the suppression of intralysosomal proteolysis

Suramin that accumulates in rat liver Kupffer cell lysosomes and inhibits the intralysosomal proteolysis was used to suppress the functional activity of these particles during liver damage (acute CCl4 hepatitis). Polyvinylpyrrolidone that does not disturb protein catabolism in liver lysosomes was employed for reference. According to the characteristic changes in lysosomes induced by suramin (inhibition of acid phosphatase, decrease of the rate of the intralysosomal proteolysis in the liver) and PVP the damaged liver was able to accumulate the lysosomotropic substances under study. Suramin aggravated liver damage and increased the lysosomal labilization, whereas PVP exhibited the protective action. The unfavourable effect of suramin may be linked with the suppression of catabolism of Kupffer cell lysosomes. The data obtained suggest the lack of safety of using the inhibitors of intralysosomal proteolysis in patients with acute hepatitis.     

Suramin induced ceramide accumulation leads to apoptotic cell death in dorsal root ganglion neurons

Suramin is an experimental antineoplastic agent that is currently being tested in clinical trials for a number of human cancers. In previous clinical trials, it has been noted that a significant percentage of patients treated with suramin develop a peripheral neuropathy. Both the cytotoxic (chemotherapeutic) and neurotoxic mechanisms of action of this compound are unknown. Evidence presented in this study suggests that both effects may be due to extensive disruption in glycolipid transport and/or metabolism. Suramin treated dorsal root ganglion cultures revealed an accumulation of the GM1 ganglioside and ceramide. Exposure of cultures to suramin, a cell permeable ceramide analog, or sphingomyelinase lead to apoptotic cell death demonstrated by electron microscopy, bis-benzimide staining and DNA laddering on gel electrophoresis. Furthermore, a significant increase in intracellular ceramide preceded cell death in suramin treated neurons. We propose that suramin induced ceramide accumulation within neurons leads to apoptotic cell death.