A combinatorial approach for targeted delivery using small molecules and reversible masking to bypass nonspecific uptake in vivo.

We have developed a multi-disciplinary approach combining molecular biology, delivery technology, combinatorial chemistry and reversible masking to create improved systemic, targeted delivery of plasmid DNA while avoiding nonspecific uptake in vivo. We initially used a well-characterized model targeting the asialolglycoprotein receptor in the liver. Using our bilamellar invaginated vesicle (BIV) liposomal delivery system with reversible masking, we increased expression in the liver by 76-fold, nearly equaling expression in first-pass organs using non-targeted complexes, with no expression in other organs. The same technology was then applied to efficiently target delivery to a human tumor microenvironment model. We achieved efficient, targeted delivery by attachment of specific targeting ligands to the surface of our BIV complexes in conjunction with reversible masking to bypass nonspecific tissues and organs. We identified ligands that target a human tumor microenvironment created in vitro by co-culturing primary human endothelial cells with human lung or pancreatic cancer cells. The model was confirmed by increased expression of tumor endothelial phenotypes including CD31 and vascular endothelial growth factor-A, and prolonged survival of endothelial capillary-like structures. The co-cultures were used for high-throughput screening of a specialized small molecule library to identify ligands specific for human tumor-associated endothelial cells in vitro. We identified small molecules that enhanced the transfection efficiency of tumor-associated endothelial cells, but not normal human endothelial cells or cancer cells. Intravenous (i.v.) injection of our targeted, reversibly masked complexes into mice, bearing human pancreatic tumor and endothelial cells, specifically increased transfection to this tumor microenvironment approximately 200-fold. Efficacy studies using our optimized targeted delivery of a plasmid encoding thrombospondin-1 eliminated tumors completely after five i.v. injections administered once every week.

Apolipoprotein E (ApoE), a novel heparin-binding protein inhibits the development of Kaposi's sarcoma-like lesions in BALB/c nu/nu mice.

Recombinant apolipoprotein E-3 (ApoE-3), expressed in Escherichia coli, was purified and used in an in vitro and an in vivo model system for acquired immunodeficiency syndrome-associated Kaposi's sarcoma (AIDS-KS). This protein blocked cell proliferation and chemotaxis of AIDS-KS cells in response to activated lymphocyte conditioned medium (AL-CM) and oncostatin M (OSM). ApoE-3 also inhibited the formation of neoangiogenic lesions induced in BALB/c nu/nu mice by AIDS-KS cells. These findings represent a novel and potentially less toxic therapeutic approach for the treatment of AIDS-KS.

Platelet thrombospondin mediates attachment and spreading of human melanoma cells.

Human platelet thrombospondin adsorbed on plastic promotes attachment and spreading of human G361 melanoma cells. Attachment is rapid, and spreading is maximal by 90 min with 60-90% of the attached cells spread. In contrast, thrombospondin promotes attachment but not spreading of human C32 melanoma cells, which attach and spread only on laminin substrates. The specificity of these interactions and the regions of the thrombospondin molecule involved in attachment and spreading were examined using proteolytic fragments of thrombospondin and by inhibition studies. The sulfated fucan, fucoidan, and monoclonal antibody A2.5, which is directed against the heparin-binding domain of thrombospondin, selectively inhibit spreading but only weakly inhibit attachment. Monoclonal antibodies against some other domains of thrombospondin, however, are potent inhibitors of attachment. The amino-terminal heparin-binding domain of thrombospondin does not promote attachment. Large fragments lacking the heparin-binding domain support attachment but not spreading of G361 cells. Attachment activity is lost following removal of the 18-kD carboxyl-terminal domain. These results suggest that at least two melanoma ligands are involved in cell attachment and spreading on thrombospondin. The carboxyl-terminal region and perhaps other regions of the molecule bind to receptor(s) on the melanoma surface that promote initial attachment but not cell spreading. Interaction of the heparin-binding domain with sulfated glycoconjugates on melanoma surface proteoglycans and/or sulfated glycolipids mediates spreading. Monoclonal antibodies A2.5 and C6.7 also reverse spreading of G361 cells growing on glass culture substrates, suggesting that binding to thrombospondin mediates attachment of these melanoma cells in culture.

Thrombospondin-induced tumor cell migration: haptotaxis and chemotaxis are mediated by different molecular domains.

Thrombospondin induces the migration of human melanoma and carcinoma cells. Using a modified Boyden chamber assay, tumor cells migrated to a gradient of soluble thrombospondin (chemotaxis). Checkerboard analysis indicated that directional migration was induced 27-fold greater than stimulation of random motility. Tumor cells also migrated in a dose-dependent manner to a gradient of substratum-bound thrombospondin (haptotaxis). A series of human melanoma and carcinoma cells were compared for their relative motility stimulation by thrombospondin haptotaxis vs. chemotaxis. Some cell lines exhibited a stronger haptotactic response compared to their chemotactic response while other lines exhibited little or no migration response to thrombospondin. Human A2058 melanoma cells which exhibit a strong haptotactic and chemotactic response to thrombospondin were used to study the structural domains of thrombospondin required for the response. Monoclonal antibody C6.7, which binds to the COOH-terminal region of thrombospondin, inhibited haptotaxis in a dose-dependent optimal manner. C6.7 had no significant effect on thrombospondin chemotaxis. In contrast, monoclonal antibody A2.5, heparin, and fucoidan, which bind to the NH2-terminal heparin-binding domain of thrombospondin, inhibited thrombospondin chemotaxis but not haptotaxis. Monoclonal antibody A6.1 directed against the internal core region of thrombospondin had no significant effect on haptotaxis or chemotaxis. Synthetic peptides GRGDS (50 micrograms/ml), but not GRGES, blocked tumor cell haptotaxis on fibronectin, but had minimal effect on thrombospondin or laminin haptotaxis. The 140-kD fragment of thrombospondin lacking the heparin-binding amino-terminal region retained the property to fully mediate haptotaxis but not chemotaxis. When the COOH region of the 140-kD fragment, containing the C6.7-binding site, was cleaved off, the resulting 120-kD fragment (which retains the RGDA sequence) failed to induce haptotaxis. Separate structural domains of thrombospondin are therefore required for tumor cell haptotaxis vs. chemotaxis. This may have implications during hematogenous cancer metastases formation.

Inhibition of fibronectin binding and fibronectin-mediated cell adhesion to collagen by a peptide from the second type I repeat of thrombospondin.

The platelet and extracellular matrix glycoprotein thrombospondin interacts with various types of cells as both a positive and negative modulator of cell adhesion, motility, and proliferation. These effects may be mediated by binding of thrombospondin to cell surface receptors or indirectly by binding to other extracellular matrix components. The role of peptide sequences from the type I repeats of thrombospondin in its interaction with fibronectin were investigated. Fibronectin bound specifically to the peptide Gly-Gly-Trp-Ser-His-Trp from the second type I repeat of thrombospondin but not to the corresponding peptides from the first or third repeats or flanking sequences from the second repeat. The two Trp residues and the His residue were essential for binding, and the two Gly residues enhanced the affinity of binding. Binding of the peptide and intact thrombospondin to fibronectin were inhibited by the gelatin-binding domain of fibronectin. The peptide specifically inhibited binding of fibronectin to gelatin or type I collagen and inhibited fibronectin-mediated adhesion of breast carcinoma and melanoma cells to gelatin or type I collagen substrates but not direct adhesion of the cells to fibronectin, which was inhibited by the peptide Gly-Arg-Gly-Asp-Ser. Thus, the fibronectin-binding thrombospondin peptide Gly-Gly-Trp-Ser-His-Trp is a selective inhibitor of fibronectin-mediated interactions of cells with collagen in the extracellular matrix.

Type I collagen is a molecular target for inhibition of angiogenesis by endogenous thrombospondin-1.

Three-dimensional explant cultures of muscle tissue were used to characterize secreted proteins regulated by endogenous levels of the angiogenesis modulator thrombospondin (TSP)-1. Explants from TSP1 null mice exhibit enhanced neovascularization associated with increased endothelial outgrowth but decreased outgrowth of perivascular smooth muscle cells . The absence of endogenous TSP1 did not diminish activation of latent transforming growth factor-beta and moderately decreased matrix metalloproteinase levels. However, significant changes in other secreted proteins were observed. Endogenous TSP1 decreased mRNA levels for collagens Ialpha1, Ialpha2, and IIIalpha1 and laminin alpha4 and increased collagen IValpha1 mRNA expression. Endogenous TSP1 also decreased the level of type I collagen protein produced by the vascular outgrowths. Collagens Ialpha1, Ialpha2, and IIIalpha1 are known tumor endothelial markers, suggesting that TSP1 coordinately regulates a set of extracellular matrix genes that reverse the angiogenic switch. Suppression of collagen Ialpha1 or Ialpha2 mRNAs using antisense morpholinos inhibited outgrowth in TSP1 null explants and proliferation of TSP1 null endothelial cells, indicating that type I collagen synthesis is limiting for this neovascularization response.

Modulation of angiogenesis by dithiolethione-modified NSAIDs and valproic acid.

BACKGROUND AND PURPOSE: Angiogenesis involves multiple signaling pathways that must be considered when developing agents to modulate pathological angiogenesis. Because both cyclooxygenase inhibitors and dithioles have demonstrated anti-angiogenic properties, we investigated the activities of a new class of anti-inflammatory drugs containing dithiolethione moieties (S-NSAIDs) and S-valproate. EXPERIMENTAL APPROACH: Anti-angiogenic activities of S-NSAIDS, S-valproate, and the respective parent compounds were assessed using umbilical vein endothelial cells, muscle and tumor tissue explant angiogenesis assays, and developmental angiogenesis in Fli:EGFP transgenic zebrafish embryos. KEY RESULTS: Dithiolethione derivatives of diclofenac, valproate, and sulindac inhibited endothelial cell proliferation and induced Ser(78) phosphorylation of hsp27, a known molecular target of anti-angiogenic signaling. The parent drugs lacked this activity, but dithiolethiones were active at comparable concentrations. Although dithiolethiones can potentially release hydrogen sulphide, NaSH did not reproduce some activities of the S-NSAIDs, indicating that the dithioles regulate angiogenesis through mechanisms other than release of H(2)S. In contrast to the parent drugs, S-NSAIDs, S-valproate, NaSH, and dithiolethiones were potent inhibitors of angiogenic responses in muscle and HT29 tumor explants assessed by 3-dimensional collagen matrix assays. Dithiolethiones and valproic acid were also potent inhibitors of developmental angiogenesis in zebrafish embryos, but the S-NSAIDs, remarkably, lacked this activity. CONCLUSIONS AND IMPLICATION: S-NSAIDs and S-valproate have potent anti-angiogenic activities mediated by their dithiole moieties. The novel properties of S-NSAIDs and S-valproate to inhibit pathological versus developmental angiogenesis suggest that these agents may have a role in cancer treatment.

Improved DNA: liposome complexes for increased systemic delivery and gene expression.

To increase cationic liposome-mediated intravenous DNA delivery extruded DOTAP:cholesterol liposomes were used to form complexes with DNA, resulting in enhanced expression of the chloramphenicol acetyltransferase gene in most tissues examined. The DNA:liposome ratio, and mild sonication, heating, and extrusion steps used for liposome preparation were crucial for improved systemic delivery. Size fractionation studies showed that maximal gene expression was produced by a homogeneous population of DNA:liposome complexes between 200 to 450 nm in size. Cryo-electron microscopy examination demonstrates that the DNA:liposome complexes have a novel morphology, and that the DNA is condensed on the interior of invaginated liposomes between two lipid bilayers. This structure could account for the high efficiency of gene delivery in vivo and for the broad tissue distribution of the DNA:liposome complexes. Ligands can be placed on the outside of this structure to provide for targeted gene delivery.

Cell contact-dependent activation of alpha3beta1 integrin modulates endothelial cell responses to thrombospondin-1.

Thrombospondin-1 (TSP1) can inhibit angiogenesis by interacting with endothelial cell CD36 or proteoglycan receptors. We have now identified alpha3beta1 integrin as an additional receptor for TSP1 that modulates angiogenesis and the in vitro behavior of endothelial cells. Recognition of TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 by normal endothelial cells is induced after loss of cell-cell contact or ligation of CD98. Although confluent endothelial cells do not spread on a TSP1 substrate, alpha3beta1 integrin mediates efficient spreading on TSP1 substrates of endothelial cells deprived of cell-cell contact or vascular endothelial cadherin signaling. Activation of this integrin is independent of proliferation, but ligation of the alpha3beta1 integrin modulates endothelial cell proliferation. In solution, both intact TSP1 and the alpha3beta1 integrin-binding peptide from TSP1 inhibit proliferation of sparse endothelial cell cultures independent of their CD36 expression. However, TSP1 or the same peptide immobilized on the substratum promotes their proliferation. The TSP1 peptide, when added in solution, specifically inhibits endothelial cell migration and inhibits angiogenesis in the chick chorioallantoic membrane, whereas a fragment of TSP1 containing this sequence stimulates angiogenesis. Therefore, recognition of immobilized TSP1 by alpha3beta1 integrin may stimulate endothelial cell proliferation and angiogenesis. Peptides that inhibit this interaction are a novel class of angiogenesis inhibitors.

Interactions of thrombospondin with sulfated glycolipids and proteoglycans of human melanoma cells.

Human melanoma cell spreading on thrombospondin substrates and chemotaxis in a gradient of soluble thrombospondin requires the amino-terminal heparin/sulfatide-binding domain of thrombospondin. Some melanoma cell lines attach but do not spread or respond in chemotaxis assays. Sulfated glycoconjugates produced by melanoma cells that could mediate these activities were identified by metabolic labeling with [35S] sulfate and tested for their ability to bind thrombospondin. Heparan sulfate proteoglycans that bind thrombospondin are made by both spreading and non-spreading melanoma cell lines. Thrombospondin binds with high affinity to a high molecular weight heparan sulfate proteoglycan, but not to the major chondroitin sulfate. The active heparan sulfate proteoglycan can be partially purified by affinity chromatography on thrombospondin-agarose or hydrophobic interaction with octyl-Sepharose. Thrombospondin binding requires the amino-terminal domain and is inhibited by monoclonal antibody A2.5 or fucoidan. Binding activity is lost following degradation of the proteoglycan with heparatinase or nitrous acid. [35S]Sulfate labels several melanoma cell glycolipids including galactosylceramide-I3-sulfate, lactosyl ceramide-II3-sulfate, and sulfated glucuronosylparagloboside. The latter glycolipid was detected in three cell lines that spread on thrombospondin but not in the nonspreading C32 melanoma cells. Thrombospondin binds to the isolated glycolipid, and the glycolipid and an antibody to this structure inhibit cell spreading on thrombospondin substrates. Thus, the presence of glycoconjugates with terminal nonreducing glucuronosyl 3-sulfate correlates with melanoma cell spreading on thrombospondin, whereas expression of heparan sulfate proteoglycans that bind thrombospondin does not.

Suramin inhibits laminin- and thrombospondin-mediated melanoma cell adhesion and migration and binding of these adhesive proteins to sulfatide.

Suramin is a polysulfonated drug with several biological activities including inhibition of binding of some growth factors to cells, inhibition of tumor cell growth, and of glycosaminoglycan metabolism. We report here that suramin also inhibits binding of the adhesive glycoproteins, thrombospondin and laminin, to immobilized sulfatide with 50% inhibitory doses of 220 and 470 micrograms/ml, respectively. Sulfated glycoconjugates on melanoma cells mediate spreading on thrombospondin by binding to the amino-terminal heparin- and sulfatide-binding domain. This domain is also required for chemotaxis on thrombospondin. We therefore examined the effect of suramin on human melanoma cell spreading and migration. Suramin at 50-400 micrograms/ml specifically inhibited G361 melanoma cell spreading on thrombospondin without affecting cell attachment. Suramin also inhibited spreading of A2058 melanoma cells on thrombospondin and laminin and partially inhibited cell attachment. However, suramin had no effect on G361 or A2058 cell attachment or spreading on fibronectin. Chemotaxis of A2058 and G361 melanoma cells to thrombospondin and laminin were also specifically inhibited by suramin, as was haptotaxis of A2058 melanoma cells to laminin. However, suramin only weakly inhibited haptotaxis of G361 melanoma cells to thrombospondin, which is not mediated by the amino-terminal domain, and did not inhibit haptotaxis to fibronectin. These results suggest a new mechanism for the observed antitumor activity of suramin based on its ability to inhibit interactions of tumor cells with laminin or thrombospondin in the extracellular matrix.

Laminin-dependent and laminin-independent adhesion of human melanoma cells to sulfatides.

Sulfatides (galactosylceramide-I3-sulfate) but not neutral glycolipids or gangliosides adsorbed on plastic promote adhesion of the human melanoma cell line G361. Direct adhesion of G361 cells requires densities of sulfatide greater than 1 pmol/mm2. In the presence of laminin, however, specific adhesion of G361 cells to sulfatide or seminolipid (galactosylalkylacyl-glycerol-I3-sulfate) but not to other lipids is strongly stimulated and requires only 25 fmol/mm2 of adsorbed lipid. The effects of laminin and sulfatide on adhesion are synergistic, suggesting that laminin is mediating adhesion by cross-linking receptors on the melanoma cell surface to sulfatide adsorbed on the plastic. Although thrombospondin binds to sulfatides and G361 cells, it does not enhance, but rather inhibits direct and laminin-dependent G361 cell adhesion to sulfatide. In contrast, C32 melanoma cells also adhere specifically to sulfatide, but adhesion of these cells is not enhanced by laminin or inhibited by antibodies to laminin that block laminin-dependent adhesion of G361 cells. Thrombospondin is a potent inhibitor of C32 cell adhesion to sulfatide. Fucoidan, which inhibits laminin binding to sulfatide, inhibits laminin-dependent adhesion of G361 cells by 50% at 0.2 micrograms/ml. Several other tumor cell lines also attach directly on sulfatide-coated surfaces. Laminin stimulates adhesion to sulfatide of three of the six cell lines tested. The ability of laminin to promote adhesion of tumor cells to sulfatide suggests that binding to sulfatide could participate in laminin-mediated cell-cell adhesion. Thus, many tumor cell lines can attach on sulfatide substrates using endogenous sulfatide binding proteins, and in some cells laminin but not thrombospondin can promote tumor cell adhesion to sulfatide.

Thrombospondin 1 and type I repeat peptides of thrombospondin 1 specifically induce apoptosis of endothelial cells.

Thrombospondin 1 (TSP1) inhibits angiogenesis and modulates endothelial cell adhesion, motility, and growth. The antiproliferative activity of TSP1 is mimicked by synthetic peptides derived from the type I repeats of TSP1 that antagonize fibroblast growth factor 2 and activate latent transforming growth factor beta. These TSP1 analogues induced programmed cell death in bovine aortic endothelial cells based on morphological changes, assessment of DNA fragmentation, and internucleosomal DNA cleavage. Intact TSP1 also induced DNA fragmentation. The endothelial cell response was specific because no DNA fragmentation was induced in MDA-MB-435S breast carcinoma cells, although TSP1 and the peptide conjugates inhibited the growth of both cell types. Apoptosis did not depend on activation of latent transforming growth factor beta because peptides lacking the activating sequence RFK were active. Apoptosis was not sensitive to inhibitors of ceramide generation but was inhibited by the phosphatase inhibitor vanadate. Induction of DNA fragmentation by the peptides was decreased when endothelial cell cultures reached confluence. Growth of the cells on a fibronectin substrate also suppressed induction of apoptosis by TSP1 or the peptides. Differential sensitivities to kinase inhibitors suggest that apoptosis and inhibition of proliferation are mediated by distinct signal transduction pathways. These results demonstrate that induction of apoptosis by the TSP1 analogues is not a general cytotoxic effect and is conditional on a lack of strong survival-promoting signals, such as those provided by a fibronectin matrix. The antitumor activity of TSP1 may therefore result from an increased sensitivity to apoptosis in endothelial cells adjacent to a provisional matrix during formation of vascular beds in tumors expressing TSP1.

Thrombospondin-1 promotes alpha3beta1 integrin-mediated adhesion and neurite-like outgrowth and inhibits proliferation of small cell lung carcinoma cells.

Although human small cell lung carcinoma (SCLC) cell lines are typically anchorage-independent and do not attach on most extracellular matrix proteins, OH-1, and several other SCLC cell lines attached on substrates coated with thrombospondin-1 (TSP1). SCLC cells grew long-term as adherent cells on a TSP1-coated substrate. Adhesion of SCLC cells on TSP1 was inhibited by heparin, function-blocking antibodies recognizing alpha3 or beta1 integrin subunits, and by soluble alpha3beta1 integrin ligands. SCLC cells extended neurite-like processes on a TSP1 substrate, which was also mediated by alpha3beta1 integrin. Process formation on a TSP1 substrate was specifically stimulated by epidermal growth factor and somatostatin. Adhesion on TSP1 weakly inhibited SCLC cell proliferation, but this inhibition was strongly enhanced in the presence of epidermal growth factor. TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 also inhibited proliferation when added in solution. High-affinity binding of 125I-labeled TSP1 to OH-1 cells was heparin-dependent and may be mediated by sulfated glycolipids, which are the major sulfated glycoconjugates synthesized by these cells. Synthesis or secretion of TSP1 by SCLC cells could not be detected. On the basis of these results, the alpha3beta1 integrin and sulfated glycolipids cooperate to mediate adhesion of SCLC cells on TSP1. Interaction with TSP1 through this integrin inhibits growth and induces neurotypic differentiation, which suggests that this response to TSP1 may be exploited to inhibit the progression of SCLC.

Transfection of thrombospondin 1 complementary DNA into a human breast carcinoma cell line reduces primary tumor growth, metastatic potential, and angiogenesis.

Previous studies demonstrated that metastatic MDA-MB-435 breast carcinoma cells synthesized and secreted less of the extracellular matrix protein thrombospondin 1 (TSP1) than nonmetastatic breast carcinoma cell lines, a trend also observed for melanoma and lung carcinoma cell lines. To directly examine the effect of tumor cell TSP1 expression on tumor growth and metastasis. MDA-MB-435 cells were transfected with full length THBS-1 cDNA linked to a constitutive cytomegalovirus promoter, or with the cytomegalovirus vector alone. Injection of transfected clones that overexpressed TSP1 into the mammary fat pad of nude mice resulted in a dose-dependent inhibition of primary tumor size and an inhibition of spontaneous pulmonary metastases, which occurred in 21-30% of THBS-1 transfectants compared to 44-49% of controls (P = 0.007). An additional clone was identified that overexpressed a COOH-terminally truncated TSP1. This clone produced larger primary tumors and an increase in the occurrence of metastases relative to control transfectants, suggesting the participation of a previously understudied region of TSP1 in the regulation of tumor progression. The THBS-1 and control transfectants did not exhibit significant differences in growth, colonization, or motility in vitro. However, a relative reduction in capillary densities in primary tumors formed by the wild-type THBS-1 transfectants was observed, suggestive of an angiostatic effect. The data indicate that tumor cell production of TSP1 can exert a significant inhibitory effect on tumor progression in the MDA-MB-435 breast carcinoma cell line, which may be attributable in part to a reduction in angiogenesis.

Differential roles of protein kinase C and pertussis toxin-sensitive G-binding proteins in modulation of melanoma cell proliferation and motility by thrombospondin 1.

Thrombospondin 1 (TSP1) is an angiogenesis inhibitor that decreases tumor growth. We now report that TSP1 directly inhibits the proliferation of human melanoma cells. TSP1, peptides, and a recombinant fragment from the type I repeats, but not peptides that bind CD36 or CD47, inhibit the proliferation of A2058 melanoma cells. In contrast, chemotaxis is mediated by peptides or recombinant fragments from the procollagen, type I, type II, and cell-binding domains. The antiproliferative activity of TSP1 is mediated by a different signal transduction pathway than those mediating motility responses to the same protein. Activators of protein kinase A and protein kinase C inhibit chemotaxis but not the antiproliferative activity of TSP1, whereas the antiproliferative activity is reversed by inhibiting the tyrosine kinase or phosphatase activities. TSP1-mediated chemotaxis is partially dependent on a pertussis toxin (PT)-sensitive G-binding protein, whereas haptotaxis is not. Chemotaxis stimulated by the procollagen domain and the CD47-binding sequences from the COOH-terminal domain are also sensitive to PT, but responses to the type I and type III domains are not sensitive to PT. Residual chemotaxis to TSP1 in the presence of PT may therefore be mediated by the activities of the type I or type III repeats. Thus, TSP1 elicits several intracellular signals in melanoma cells that result from interactions with several domains of this protein and differentially affect growth and motility.

Dietary fat overcomes the protective activity of thrombospondin-1 signaling in the Apc(Min/+) model of colon cancer.

Thrombospondin 1 is a glycoprotein that regulates cellular phenotype through interactions with its cellular receptors and extracellular matrix-binding partners. Thrombospondin 1 locally regulates angiogenesis and inflammatory responses that contribute to colorectal carcinogenesis in Apc(Min/+) mice. The ability of thrombospondin 1 to regulate responses of cells and tissues to a variety of stresses suggested that loss of thrombospondin 1 may also have broader systemic effects on metabolism to modulate carcinogenesis. Apc(Min/+):Thbs1(-/-) mice exhibited decreased survival and higher tumor multiplicities in the small and large intestine relative to Apc(Min/+) mice when fed a low (5%) fat western diet. However, the protective effect of endogenous thrombospondin 1 was lost when the mice were fed a western diet containing 21% fat. Biochemical profiles of liver tissue identified systemic metabolic changes accompanying the effects of thrombospondin 1 and dietary lipid intake on tumorigenesis. A high-fat western diet differentially regulated elements of amino acid, energy and lipid metabolism in Apc(Min/+):Thbs1(-/-) mice relative to Apc(Min/+):Thbs1(+/+)mice. Metabolic changes in ketone body and tricarboxylic acid cycle intermediates indicate functional interactions between Apc and thrombospondin 1 signaling that control mitochondrial function. The cumulative diet-dependent differential changes observed in Apc(Min/+):Thbs1(-/-) versus Apc(Min/+) mice include altered amino acid and lipid metabolism, mitochondrial dysfunction, eicosanoids and ketone body formation. This metabolic profile suggests that the protective role of thrombospondin 1 to decrease adenoma formation in Apc(Min/+) mice results in part from improved mitochondrial function.

Inhibition of angiogenesis by thrombospondin-1 is mediated by 2 independent regions within the type 1 repeats.

BACKGROUND: Suppression of tumor growth by thrombospondin-1 (TSP-1) has been associated with its ability to inhibit neovascularization. The antiangiogenic activity of TSP-1, as defined by cornea pocket assays, was previously mapped to the amino-terminal portion of the protein within the procollagen region and the type 1 repeats. METHODS AND RESULTS: We evaluated the specificity and efficacy of different regions of TSP-1 using recombinant fragments of the protein on chorioallantoic membrane (CAM) angiogenesis and endothelial cell proliferation assays. In both assays, fragments containing the second and third type 1 repeats but not the procollagen region inhibited angiogenesis and endothelial cell proliferation. To further define the sequences responsible for the angiostatic effect of TSP-1, we used synthetic peptides. The CAM assay defined 2 sequences that independently suppressed angiogenesis. The amino-terminal end of the type 1 repeats showed higher potency for inhibiting angiogenesis driven by basic fibroblast growth factor (FGF-2), whereas the second region equally blocked angiogenesis driven by either FGF-2 or vascular endothelial growth factor (VEGF). Modifications of the active peptides revealed the specific amino acids required for the inhibitory response. One sequence included the conserved tryptophan residues in the amino-terminal end of the second and third type 1 repeats, and the other involved the amino acids that follow the CSVTCG sequence in the carboxy-terminus of these repeats. Both inhibition in the CAM assay and inhibition of breast tumor xenograft growth in nude mice were independent of the TGF-beta-activating sequence located in the second type 1 repeat. CONCLUSIONS: These results indicate that the type 1 repeats of TSP-1 contain 2 subdomains that may independently inhibit neovascularization. They also identify 2 independent pathways by which TSP-1 can block FGF-2 and VEGF angiogenic signals on endothelial cells.

Semenogelins are ectopically expressed in small cell lung carcinoma.

Two proteins recovered from cell surface adhesion complexes in a small cell lung carcinoma (SCLC) cell line were identified as fragments of the seminal plasma proteins semenogelin I and semenogelin II. Association of both proteins with the adhesion complexes was induced by epidermal growth factor. Expression of semenogelins was previously thought to be highly specific to seminal vesicles, but Western blot analysis demonstrated that semenogelin II is widely expressed in SCLC cell lines and occasionally in other malignant cell lines. Although semenogelin expression is normally restricted to males, two SCLC cell lines from female patients were also positive for semenogelin II expression. Immunohistochemical analysis demonstrated diffuse expression of semenogelins in 12 of 13 SCLC tumors and focal expression in a minority of lung squamous and adenocarcinomas. Semenogelins were secreted into the medium by cultured SCLC cells, which suggested that these proteins may be useful markers for detecting residual tumor burden or recurrence of SCLC after treatment.

Treatment of experimental brain tumors with trombospondin-1 derived peptides: an in vivo imaging study.

Antiangiogenic and antiproliferative effects of synthetic D-reverse peptides derived from the type 1 repeats of thrombospondin (TSP1) were studied in rodent C6 glioma and 9L gliosarcomas. To directly measure tumor size and vascular parameters, we employed in vivo magnetic resonance (MR) imaging and corroborated results by traditional morphometric tissue analysis. Rats bearing either C6 or 9L tumors were treated with TSP1-derived peptide (D-reverse amKRFKQDGGWSHWSPWSSac, n=13) or a control peptide (D-reverse amKRAKQAGGASHASPASSac, n=12) at 10 mg/kg, administered either intravenously or through subcutaneous miniosmotic pumps starting 10 days after tumor implantation. Eleven days later, the effect of peptide treatment was evaluated. TSP1 peptide-treated 9L tumors (50.7+/-44.2 mm3, n=7) and C6 tumors (41.3+/-34.2 mm3, n=6) were significantly smaller than tumors treated with control peptide (9L: 215.7+/-67.8 mm3, n=6; C6: 184.2+/-105.2 mm3, n=6). In contrast, the in vivo vascular volume fraction, the mean vascular area (determined by microscopy), and the microvascular density of tumors were not significantly different in any of the experimental groups. In cell culture, TSP1, and the amKRFKQDGGWSHWSPWSSac peptide showed antiproliferative effects against C6 with an IC of 45 nM for TSP1. These results indicate that TSP1-derived peptides retard brain tumor growth presumably as a result of slower de novo blood vessel formation and synergistic direct antiproliferative effects on tumor cells. We also show that in vivo MR imaging can be used to assess treatment efficacy of novel antiangiogenic drugs non-invasively, which has obvious implications for clinical trials.