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 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.     

Cellular expression of bone-related proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures

The regulation of cell surface fibroblast growth factor (FGF) receptors during the differentiation of F9 teratocarcinoma cells was investigated. The capacity of F9 cells to bind ¹²⁵I-basic FGF (FGF-2) increased upon induction of differentiation with dibutyryl cAMP and retinoic acid. No change in binding capacity was observed in the first 24 h after addition of differentiating agents, but a sixfold increase in binding capacity was observed after 48 h and a fivefold increase after 72 h. Scatchard analysis of the binding data indicated that the increased binding of ¹²⁵I-FGF-2 was due to an increase in the number of receptors with no change in their affinity. When ¹²⁵I-FGF-2 was cross-linked to cell surface receptors, an increase in FGF-2-receptor complexes with molecular weights of 140,000–160,000 was also observed in the differentiated F9 cells. Undifferentiated F9 cells are known to secrete FGF-4 and cease expression of this molecule upon differentiation. To determine whether the low level of receptors in undifferentiated cells might be related to their production of FGF ligands, the ability of suramin, a drug that can disrupt FGF-receptor interactions, to modulate receptor number on F9 cells was investigated. Suramin treatment increased ¹²⁵I-FGF-2 binding capacity of undifferentiated F9 cells threefold but had little effect on the binding capacity of differentiated cells. In addition, antibodies to FGF-4 increased the ¹²⁵I-FGF-2 binding capacity of undifferentiated F9 cells by 58%. These results suggest that undifferentiated F9 cells might be responding in an autocrine manner to their own FGF ligands resulting in downregulation of cell surface FGF receptors. The increased number of receptors observed in differentiated cells may partly result from the decreased production of FGF ligands by these cells. © 1994 Wiley-Liss, Inc.     

Androgen receptor, testosterone uptake and karyotype in androgen-dependent mouse tumor (SC 115) and its androgen-independent subline (CS 2)

Content of androgen receptor, retention of injected testosterone and karyotype of SC 115, androgen-dependent tumor, were compared with those of CS 2, an androgen-independent subline derived from SC 115. Although Bmax was less than that of SC 115, androgen receptor was present in the cytosol and the nuclear extract from CS 2. To examine the ability for androgen retention, a large amount of testosterone was injected into tumor-bearing mice, and the amount of androgen in the crude nuclear and postnuclear fractions of tumors was compared. In both fractions, retention of injected androgen was higher in the SC 115 than in the CS 2. Since most of the injected testosterone was not metabolized in the tissues and the injection of testosterone 5 alpha-reductase inhibitor showed no significant influence on the growth rate of the SC 115, intracellular active androgen was assumed to be testosterone in these tumor cells. As the CS 2 was tetraploid, the androgen independency of the CS 2 seems to be related to chromosomal changes.     

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 Induces Phosphorylation of the High-Affinity Nerve Growth Factor Receptor in PC12 Cells and Dorsal Root Ganglion Neurons

Abstract: Suramin is a polysulfonated naphthylurea with demonstrated antineoplastic activity. Toxicity includes adrenal insufficiency and peripheral neuropathy. Although the mechanism of antitumor activity is unknown, inhibition of binding of growth factors to their receptors has been suggested. Growth factors inhibited by suramin include platelet-derived growth factor, fibroblast growth factor, transforming growth factor, epidermal growth factor, insulin-like growth factor, and nerve growth factor (NGF). In these studies, suramin was shown to be cytotoxic to PC12 cells in a dose-dependent manner. At lower doses and in surviving cells, we observed the induction of neurite outgrowth. To determine the mechanism of suramin-induced neurite outgrowth, PC12 cells were exposed to suramin and/or NGF for various time periods and treated cells were analyzed, by western blot analysis, for expression of tyrosine phosphoproteins. There was a similarity in the pattern of tyrosine-phosphorylated proteins in PC12 cells stimulated with suramin or NGF. Of particular interest was the rapid phosphorylation (by 1 min) of the high-affinity NGF (TrkA) receptor. Activation of other members of the signal-transduction cascade (Shc, p21 ^ras , Raf-1, ERK-1) revealed similar phosphorylation levels induced by suramin and NGF. Parallel studies were performed in rat dorsal root ganglion cultures; suramin potentiated neurite outgrowth and activated the NGF receptor on these cells. This finding of specific patterns of tyrosine phosphorylation of cellular proteins in response to suramin treatment demonstrated that suramin is a partial agonist for the NGF receptor in both PC12 cells and dorsal root ganglion neurons.     

Heparin inhibits autocrine stimulation but not fibroblast growth factor stimulation of cell proliferation of androgen-responsive Shionogi carcinoma 115.

An androgen-responsive cloned cell line (SC-3) derived from Shionogi carcinoma 115 (SC115) has been shown to secrete fibroblast growth factor (FGF)-like peptide in response to androgen, which binds to FGF receptor and promotes the proliferation of SC-3 cells in an autocrine mechanism. Since the androgen-induced autocrine factor has a property to bind heparin, we examined the effects of heparin on the growth of SC-3 cells. Heparin was found to exhibit significant inhibition of testosterone-induced growth in a concentration-dependent manner: Approximately 50% inhibition was found at a concentration of 0.1 micrograms/ml. DNA synthesis of SC-3 cells induced by testosterone was also inhibited strongly by heparin, and less strongly by heparan sulfate and dermatan sulfate. Proliferation of SC-3 cells induced by acidic (a) or basic (b) FGF appeared not to be modulated by heparin. In contrast, heparin efficiently blocked DNA synthesis stimulated with androgen-induced growth factor in the conditioned medium from testosterone-treated cells. These results indicate that heparin inhibits autocrine loop in SC-3 cells induced by androgen. Thus, the autocrine growth factor possesses a different characteristic from aFGF and bFGF in that its bioactivities are negatively modulated by the glycosaminoglycan.     

The Beneficial Effect of Suramin on Monocrotaline-Induced Pulmonary Hypertension in Rats

Background

Pulmonary hypertension (PH) is a progressive disorder characterized by an increase in pulmonary artery pressure and structural changes in the pulmonary vasculature. Several observations indicate that growth factors play a key role in PH by modulating pulmonary artery smooth muscle cell (PA-SMC) function. In rats, established monocrotaline-induced PH (MCT-PH) can be reversed by blocking platelet-derived growth factor receptors (PDGF-R), epidermal growth factor receptors (EGF-R), or fibroblast growth factor receptors (FGF-R). All these receptors belong to the receptor tyrosine kinase (RTK) family.

Methods and Results

We evaluated whether RTK blockade by the nonspecific growth factor inhibitor, suramin, reversed advanced MCT-PH in rats via its effects on growth-factor signaling pathways. We found that suramin inhibited RTK and ERK1/2 phosphorylation in cultured human PA-SMCs. Suramin inhibited PA-SMC proliferation induced by serum, PDGF, FGF2, or EGF in vitro and ex vivo. Treatment with suramin from day 1 to day 21 after monocrotaline injection attenuated PH development, as shown by lower values for pulmonary artery pressure, right ventricular hypertrophy, and distal vessel muscularization on day 21 compared to control rats. Treatment with suramin from day 21 to day 42 after monocrotaline injection reversed established PH, thereby normalizing the pulmonary artery pressure values and vessel structure. Suramin treatment suppressed PA-SMC proliferation and attenuated both the inflammatory response and the deposition of collagen.

Conclusions

RTK blockade by suramin can prevent MCT-PH and reverse established MCT-PH in rats. This study suggests that an anti-RTK strategy that targets multiple RTKs could be useful in the treatment of pulmonary hypertension.     

Understanding the mechanism of the antimitogenic activity of suramin

Fibroblast growth factors (FGFs) play crucial roles in the regulation of key cellular processes such as angiogenesis, differentiation, and tumor growth. Suramin, a polysulfonated naphthylurea, is known to be a potent inhibitor of FGF-induced angiogenesis. Using isothermal titration calorimetry, we demonstrate that human acidic fibroblast growth factor (hFGF-1) binds to suramin with high affinity in the nanomolar range. The suramin:hFGF-1 binding stoichiometry is estimated to be 2:1. Size-exclusion chromatography data reveal that suramin oligomerizes hFGF-1 to form a stable tetramer. Thermal unfolding experiments monitored by steady state fluorescence, and limited trypsin digestion analysis data suggest that suramin-induced oligomerization of hFGF-1 occurs in two steps. The first step involves the binding of suramin at specific sites on the protein. Two molecules of suramin appear to bind simultaneously to one molecule of hFGF-1. Binding of suramin possibly involves formation of solvent-exposed nonpolar surfaces in hFGF-1. In the second step, FGF appears to oligomerize through coalescence of the solvent-accessible nonpolar surfaces. Results of the NMR experiments reveal that suramin binds to residues in the heparin binding pocket as well as to residues involved in FGF receptor binding. On the basis of the results of this study, we propose a model to explain the molecular mechanism(s) underlying the antimitogenic activity of suramin. To our knowledge, this is the first study in which suramin interaction sites on FGF have been characterized.     

Loss of androgen dependency with preservation of functional androgen receptors in androgen-dependent mouse tumor (Shionogi Carcinoma 115).

Shionogi Carcinoma 115 (SC 115) is an androgen-dependent mouse tumor. Chiba Subline 2 (CS 2) is an androgen-independent subline derived from SC 115. CS 2 contains androgen receptors (AR), but is refractory to androgen and does not exhibit androgen-related responses which are observed in SC 115. In the present study the structure and function of AR in SC 115 and CS 2 are examined using cloned cells. There were no gross rearrangements or deletions in the AR genes of these cell lines when compared by Southern blot analysis with the AR gene in the mouse seminal vesicle. SC 115 and CS 2 expressed AR mRNA of normal size. When the cDNA containing DNA- and androgen-binding domains of the AR genes of both cell lines were amplified by polymerase chain reaction, no mutations were found in these regions. SC 115 and CS 2 were transfected with a plasmid containing a long terminal repeat of mouse mammary tumor virus linked to the chloramphenicol acetyltransferase (CAT) gene. Androgen stimulation of these transfectants resulted in equal elevation of CAT activity. These results indicated that the androgen-independent CS 2 contained functionally normal AR which were identical to those in the androgen-dependent parent tumor.     

Novel regulation of fibroblast growth factor 2 (FGF2)-mediated cell growth by polysialic acid

Polysialic acid (polySia) is a unique polysaccharide that modifies neural cell adhesion molecule (NCAM) spatiotemporally. Recently, we demonstrated that polySia functions as a reservoir for several neurotrophic factors and neurotransmitters. Here, we showed the direct interaction between polySia and fibroblast growth factor-2 (FGF2) by native-PAGE, gel filtration, and surface plasmon resonance. The minimum chain length of polySia required for the interaction with FGF2 was 17. Compared with heparan sulfate, a well known glycosaminoglycan capable of forming a complex with FGF2, polySia formed a larger complex with distinct properties in facilitating oligomerization of FGF2, as well as in binding to FGF receptors. In polySia-NCAM-expressing NIH-3T3 cells, which were established by transfecting cells with either of the plasmids for the expression of the polysialyltransferases ST8SiaII/STX and ST8SiaIV/PST that can polysialylate NCAM, FGF2-stimulated cell growth, but not cell survival, was inhibited. Taken together, these results suggest that polySia-NCAM might be involved in the regulation of FGF2-FGF receptor signaling through the direct binding of FGF2 in a manner distinct from heparan sulfate.     

Effects of antiandrogens on growth of androgen-dependent mouse mammary tumor (Shionogi carcinoma 115) in vivo and in vitro

Binding affinities of modified steroidal anthrasteroids, 3 beta-hydroxy-3a beta,6-dimethyl-2,3,3a,4,5,8,9,10,10a beta,11,11a beta, 11b alpha-dodecahydro-1H-cyclopenta[a]anthracene-8-one (1) and 3a beta,6-dimethyl-2,3,3a,4,5,8,9,10,10a beta,11,11a beta,11b alpha-dodecahydro-1H-cyclopenta[a]anthracene-3,8-dione (2), the steroid oxendolone and the nonsteroid AA560, for the androgen receptor (AR) of Shionogi carcinoma 115 (SC115) and their effects on the growth of SC115 were investigated in vivo and in vitro. The inhibitory effects of these compounds on testosterone 5 alpha-reductase of SC115 tissues were also measured. The relative binding affinities of these compounds were 3.17-0.03% of that of dihydrotestosterone, and their rank order was (1) greater than AA560 greater than oxendolone much greater than (2). In the presence of 10(-9) M testosterone, anthrasteroids and AA560 inhibited the growth of SC115 cells at 10(-7) M in a serum-free medium, but oxendolone did not. In the absence of testosterone, (1), (2) and oxendolone promoted cell growth at 10(-6), 10(-7) and 10(-7) M, respectively. However, AA560 nearly completely blocked cell growth at 10(-5) M. At a 2 mg daily dose for 13 days, (1) and AA560 powerfully inhibited tumor growth in castrated DS mice treated with testosterone propionate but oxendolone had almost no effect. Anthrasteroids and oxendolone showed weak but significant agonistic activity in vivo. Anthrasteroids markedly inhibited 5 alpha-reductase activity of SC115, oxendolone weakly and AA560 not at all. The remarkable antiandrogenic activities of (1) and AA560 may partially result from their higher affinities for the AR of SC115 but other yet unknown mechanisms may also contribute to these activities.     

Effect of androgen on ornithine decarboxylase activity in androgen-dependent mouse mammary tumor (Shionogi Carcinoma 115) and its androgen-independent subline (CS 2)

The activity of ornithine decarboxylase in androgen-dependent mouse mammary tumor (Shionogi Carcinoma 115) was reduced to 25% by castration of tumor-bearing mice and restored to the normal level 12 h after administration of testosterone or 5 alpha-dihydrotestosterone. Administration of estradiol-17 beta to the tumor-bearing castrated mice also stimulated the enzyme activity while progesterone and cortisol had little effect. On the other hand, the enzyme activity was affected by neither castration nor androgen injection to CS 2, which is a subline of SC 115 and completely independent of androgen for growth. The inhibition of ornithine decarboxylase activity in SC 115 by injecting alpha-difluoromethylornithine did not affect the enhancement of RNA polymerase I activity by androgen, showing independent elevation of the levels of the two enzymes by androgen.     

In vitro effect of androgen on RNA synthesis in nuclei from androgen-independent subline of Shionogi carcinoma (CS 2)

By serial transplantation of CS 1, a subline of Shionogi carcinoma SC 115, to female mice, another subline was obtained and designated CS2. The subline showed a complete loss of androgen dependency on the growth of the tumor. When male mice bearing the tumor were castrated and treated with testosterone, the activity of RNA polymerase I in isolated nuclei from the tumor hardly varied during the period of the experiments (36 h), while the activity of RNA polymerase II exhibited a transient increase (about 40%) at 6 h after the testosterone injection. The results, together with the previous ones showing 80% and 40% increases in RNA polymerase I activity at 24 h after testosterone administration in the case of SC 115 (androgen-dependent tumor) and CS 1 (less androgen-dependent tumor), respectively, indicate that the stimulation of RNA polymerase I activity by androgen in the tumor tissues is closely related to the androgen dependency on the growth of the tumors.     

Suramin inhibits death receptor-induced apoptosis in vitro and fulminant apoptotic liver damage in mice

Suramin is a polysulfonated derivative of urea and has been widely used both to treat infections and as a chemotherapeutic drug. Suramin has been shown to inhibit growth factor signaling pathways; however, its effect on apoptosis is unknown. Here we show that suramin inhibits apoptosis induced through death receptors in hepatoma and lymphoma cells. It also inhibits the proapoptotic effect of chemotherapeutic drugs. The antiapoptotic mechanism is specific to cell type and is caused by reduced activation, but not altered composition, of the death-inducing signaling complex (DISC), and by inhibition of the initiator caspases 8, 9 and 10. Suramin also shows similar effects in in vivo models: apoptotic liver damage induced by CD95 stimulation and endotoxic shock mediated by tumor-necrosis factor (TNF) are inhibited in mice, but necrotic liver damage is not inhibited in a rat model of liver transplantation. Thus, the antiapoptotic property of suramin in the liver may be therapeutically exploited.     

FGF3 attached to a phosholipid membrane anchor gains a high transforming capacity. Implications of microdomains for FGF3 cell transformation

NIH3T3 cells transformed by mouse FGF3-cDNA (DMI cells) selected for their ability to grow as anchorage-independent colonies in soft agar and in defined medium lacking growth factors exhibit a highly transformed phenotype. We have used dominant negative (DN) fibroblast growth factor (FGF) receptor 2 (FGFR2) isoforms to block the FGF response in DMI cells. When the DN-FGFR was expressed in DMI cells, their transformed phenotype can be reverted. The truncated FGFR2(IIIb), the high affinity FGFR for FGF3, is significantly more efficient at reverting the transformed phenotype as the IIIc isoform, reaffirming the notion that the affinity of the ligand to the DN-FGFR2 isoform determines the effect. Heparin or heparan sulfate displaces FGF3 from binding sites on the cell surface inhibiting the growth of DMI cells and reverts the transformed phenotype (). However, the presence of heparin is necessary to induce a mitogenic response in NIH3T3 cells when stimulated with soluble purified mouse FGF3. We have investigated the importance of cell surface binding of FGF3 for its ability to transform NIH3T3 cells by creating an FGF3 mutant anchored to the membrane via glycosylphosphatidylinositol (GPI). The GPI anchor renders the cell surface association of FGF3 independent from binding to heparan sulfate-proteoglycan of the cell surface membrane. Attachment of a GPI anchor to FGF3 also confers a much higher transforming potential to the growth factor. Even more, the purified GPI-attached FGF3 is as much transforming as the secreted protein acting in an autocrine mode. Because NIH3T3 cells do not express the high affinity tyrosine kinase FGF receptors for FGF3, these findings suggest that FGF3 attached to GPI-linked heparan sulfate-proteoglycan may have a broader biological activity as when bound to transmembrane or soluble heparan sulfate-proteoglycan.     

Heparan and chondroitin sulfate on growth plate perlecan mediate binding and delivery of FGF-2 to FGF receptors.

Fibroblast growth factor (FGF)-2 regulates chondrocyte proliferation in the growth plate. Heparan sulfate (HS) proteoglycans bind FGF-2. Perlecan, a heparan sulfate proteoglycan (HSPG) in the developing growth plate, however, contains both HS and chondroitin sulfate (CS) chains. The binding of FGF-2 to perlecan isolated from the growth plate was evaluated using cationic filtration (CAF) and immunoprecipitation (IP) assays. FGF-2 bound to perlecan in both the CAF and IP assays primarily via the HS chains on perlecan. A maximum of 123 molecules of FGF-2 was calculated to bind per molecule of perlecan. When digested with chondroitinase ABC to remove its CS chains, perlecan augmented binding of FGF-2 to the FGFR-1 and FGFR-3 receptors and also increased FGF-2 stimulation of [(3)H]-thymidine incorporation in BaF3 cells expressing these FGF receptors. These data show that growth plate perlecan binds to FGF-2 by its HS chains but can only deliver FGF-2 to FGF receptors when its CS chains are removed.     

DNA synthesis in U-2 OS human osteosarcoma cells is independent of PDGF binding to functional cell surface receptors

Previous studies have shown that suramin reveals specific PDGF binding sites on U-2 OS human osteosarcoma cells. Studies presented here indicate that U-2 OS cells pretreated with suramin internalize and degrade 125I-PDGF and respond to PDGF by increased tyrosine kinase activity and amino acid transport. However, DNA synthesis in these cells is not reduced by incubation with the PDGF blocking agent suramin and is not stimulated by exogenous PDGF. These data indicate that U-2 OS cells possess functional PDGF receptors but that high levels of DNA synthesis in these cells is unrelated to the binding of secreted PDGF to these cell surface receptors. Thus, it is unlikely that the PDGF mitogen produced by U-2 OS cells stimulates proliferation through an autocrine mechanism involving secretion and subsequent binding to PDGF receptors.