Angiocidin induces differentiation of acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is a malignant proliferative disorder in which leukemic cells fail to terminally differentiate and accumulate in the blood and bone marrow. Standard AML therapy requires intensive chemotherapy with a low rate of durable remission and is associated with significant treatment-related toxicity, especially in elderly patients. Therefore, new therapeutic options for the treatment of AML are urgently needed. We previously reported that the novel angiogenic inhibitor, angiocidin, induces differentiation of monocytes to macrophages. Here we investigate the effects of angiocidin on AML cells lines and primary AML cells. Differentiation was assessed by flow cytometry measuring the increase in expression of cell surface marker characteristic of normal macrophages. Four AML cell lines (THP-1, Mono-mac-1, HL-60 and MV4-11) and 5 of 10 primary human AML samples showed evidence of differentiation when cultured in vitro for 24 h with 10 µg/mL angiocidin. Additionally, we found that angiocidin promoted secretion of a number of cytokines from the cell lines as well as patient cells. We next evaluated the effect of angiocidin on a xenotransplanted primary human AML sample engrafted in NSG mice. We found angiocidin monotherapy reduced the human AML burden in bone marrow by 63% relative to untreated control. Interestingly, angiocidin+cytosine arabinoside (Ara-C) combination therapy reduced human AML in bone marrow by 79%. We believe the combination of in vitro data supporting the capacity of angiocidin to drive differentiation in multiple AML cell lines and primary human AML samples and its activity in a xenotransplantation model that reproduces the human disease is significant. These observations support the continued evaluation and development of angiocidin as a potential novel, non-toxic therapy for AML.

G-protein coupled receptor-associated sorting protein 1 (GASP-1), a ubiquitous tumor marker.

Using an innovative "2-D high performance liquid electrophoresis" (2-D HPLE) technology we identified that a specific fragment of G-protein coupled receptor-associated sorting protein 1 (GASP-1) was present in the sera of breast cancer patients and was over-expressed in early and late stage breast tumors (Tuszynski, G.P. et al., 2011). In this study we further investigated the significance of GASP-1 as a tumor marker by investigating the expression GASP-1 in different kinds of tumors as well as in the sera of patients with various cancers. Over expression of GASP-1 was detected in brain, pancreatic, and breast cancers as compared to their respective normal tissues as assessed by immunohistochemical staining of tissue arrays using a "peptide specific" GASP-1 antibody. We found that across these cancers, GASP-1 was expressed approximately 10 fold more in the cancer as compared to normal tissue. The increase in GASP-1 expression was also seen in hyperplastic and inflammatory lesions of breast and pancreatic cancers as compared to normal tissue. GASP-1 was primarily expressed in the tumor epithelium of the epithelial-derived cancers and in the transformed glial cells of the brain tumors. Using a sensitive "competitive ELISA" for GASP-1, we found that sera from patients with brain, liver, breast and lung cancers expressed 4-7 fold more GASP-1 peptide than sera from normal healthy individuals. These studies identify GASP-1 as a potential new serum and tumor biomarker for several cancers and suggest that GASP-1 may be a novel target for development of cancer therapeutics.

Angiocidin inhibits breast cancer proliferation through activation of epidermal growth factor receptor and nuclear factor kappa (NF-ĸB).

Angiocidin, a tumor-associated peptide, has been previously shown to inhibit tumor progression by blocking angiogenesis. We now show that angiocidin has a direct inhibitory effect on tumor cell proliferation. MDA-MB-231 breast cancer cells were inhibited from proliferating in the presence of epidermal growth factor (EGF) and angiocidin. Angiocidin transfected breast cancer cells also displayed growth inhibition in vitro and failed to develop significant tumors in mice as compared to vector controls. The anti-proliferative effect of angiocidin was reversed by treating the cells with the epidermal growth factor receptor (EGFR) inhibitor 4557W, a potent tyrosine kinase inhibitor. Consistent with these results, we found that treatment of breast cancer cells with angiocidin induced a 2.3 fold increase in EGFR tyrosine 845 phosphorylation while no change in phosphorylation was observed in the remaining 16 phosphorylation sites of EGFR and those of its family members as measured by a human EGFR phosphorylation array. Treatment of breast cancer cells with angiocidin also resulted in the activation of nuclear factor ĸB (Nf-ĸB) and the de novo up-regulation of many down-stream genes transcribed by Nf-ĸB, including cytokines, inflammatory mediators and the cell cycle inhibitor p21(waf1). Therefore, angiocidin is a peptide that not only inhibits tumor angiogenesis but also directly induces inhibition of tumor growth progression through the activation of EGFR and down-stream genes transcribed by Nf-ĸB.

G-protein coupled receptor-associated sorting protein 1 (GASP-1), a potential biomarker in breast cancer.

An innovative "2-D high performance liquid electrophoresis" (2-D HPLE) technology was used to identify serum biomarkers associated with the early stage of breast cancer in addition to other more advanced stages. 2-D HPLE is a newly developed electrophoretic technology that separates 100s of serum albumin complexes on a polyvinyl membrane based on their surface charges. Association of cancer proteins or their fragments (biomarkers) with pre-existing albumin complexes in the blood of cancer patients results in altered mobility on the membrane. Using 2-D HPLE we identified that a specific fragment of G-protein coupled receptor-associated sorting protein 1 (GASP-1) was present in the sera of patients with early stage disease but absent in sera of normal patients. GASP-1 has been shown to modulate lysosomal sorting and functional down-regulation of a variety of G-protein coupled receptors in neuronal cells. However, no reports have linked GASP-1 to breast cancer pathogenesis. GASP-1 was detected in tumor extracts of 7 cases of Stage 2 and Stage 3 breast cancers, but not in adjacent normal tissue as revealed by western blot analysis using an antibody developed against a GASP-1 peptide identified by our 2-D HPLE technology. Using this antibody, we immunohistochemically detected over-expression of GASP-1 in all of 107 cases of archived ductal breast carcinoma tumor samples, while normal adjacent breast tissue from 12 cases of ductal carcinoma showed little or no staining. Additionally, all 10 cases of metastatic breast carcinoma present in lymph nodes were positive. Strong positive GASP-1 staining was observed in all tumor tissue including ductal carcinoma in situ (DCIS) and invasive ductal carcinoma. Additionally, we observed a wide spectrum of enhanced staining of premalignant ductal epithelial cells present in benign ducts and those found in atypical ductal hyperplasia (ADH). These studies identify GASP-1 as a potential new serum and tumor biomarker for breast cancer and suggest that GASP-1 may be a novel target for the development of breast cancer therapeutics.

The interaction of angiocidin with tissue transglutaminase.

Angiocidin, a matrix bound and tumor associated protein, has been shown to inhibit tumor progression and angiogenesis. We previously demonstrated that angiocidin binds to thrombospondin-1 and alpha2beta1 integrin. We now show that angiocidin binds and is a preferred substrate for tissue transglutaminase-2 (tTgase). Angiocidin bound tTgase saturably with a Kd of 26 nM, while an angiocidin deletion mutant missing the matrix binding domain of angiocidin failed to bind tTgase. tTgase colocalized with angiocidin on endothelial cells. tTgase bound anti-angiocidin immunoprecipitates of endothelial cell lysates. Breast cancer cells expressing high levels of tTgase attached to angiocidin immobilized on tissue culture plates. Angiocidin was a preferred substrate for tTgase forming high molecular weight cross-linked multimers when treated with tTgase. Cross-linked angiocidin contained iso-peptide bonds as demonstrated by Western blotting and immunohistochemical colocalization studies using endothelial cells treated with angiocidin. Cross-linked angiocidin inhibited cell migration in contrast to monomeric angiocidin and inhibited localization of fibronectin (FN), a pro-tumorigenic matrix protein, into the extracellular matrix (ECM) of tumor and HUVE cells. Our studies provide an additional explanation for the anti-tumor activity of angiocidin suggesting that cross-linked angiocidin disrupts the tumor ECM making it less permissive for tumor growth.

Interaction of alpha9beta1 integrin with thrombospondin-1 promotes angiogenesis.

Thrombospondin-1 is a multifunctional protein interacting with several cell surface receptors including integrins. We found that it is a ligand for alpha9beta1 integrin, and has an integrin binding site within its N-terminal domain (NoC1). Interaction of thrombospondin-1 and its recombinant NoC1 domain with alpha9beta1 integrin was confirmed in ELISA and cell adhesion assays. Binding of NoC1 to cells expressing alpha9beta1 integrin activated signaling proteins such as Erk1/2 and paxillin. Blocking of this integrin by monoclonal antibody and the met-leu-asp-disintegrin inhibited dermal human microvascular endothelial cell proliferation and NoC1-induced migration of these cells. Immunohistochemical studies revealed that alpha9beta1 is expressed on microvascular endothelium in several organs including skin, lung, heart and brain. NoC1 induced neovascularization in an experimental quail chorioallantoic membrane system and Matrigel plug formation assay in mice. This proangiogenic activity of NoC1 in vivo was inhibited by alpha9beta1 inhibitors. In summary, our results revealed that alpha9beta1 integrin expressed on microvascular endothelial cells interacts with thrombospondin-1, and this interaction is involved in modulation of angiogenesis.

Thrombospondin-1 (TSP-1) up-regulates tissue inhibitor of metalloproteinase-1 (TIMP-1) production in human tumor cells: exploring the functional significance in tumor cell invasion.

Thrombospondin-1 (TSP-1), a matrix-bound adhesive glycoprotein, has been shown to modulate tumor progression. We previously demonstrated that TSP-1 up-regulates matrix metalloproteinases MMP-2 and MMP-9. Our studies suggested that the balance between MMPs and tissue inhibitors of metalloproteinases (TIMPs) is a key determinant in tumor cell invasion. We now report that TSP-1 up-regulates TIMP-1 expression in both human breast and prostate cancer cell lines. The effect of TSP-1 on TIMP-1 expression was examined in human breast adenocarcinoma cell lines (MDA-MB-231) and human prostate cancer cell lines (PC3-NI and PC3-ML) treated with exogenous TSP-1. TIMP-1 expression was also examined in TSP-1 stably transfected breast cancer cell line (MDA-MB-435). Northern and western blot analysis revealed TIMP-1 mRNA and TIMP-1 protein expression increased with increasing concentrations of TSP-1. This effect was inhibited by antibodies against the type I repeat domain of TSP-1 further suggesting that TSP-1 mediates TIMP-1 secretion. Inhibition of TSP-1 induced TIMP-1 levels increased tumor cell invasion. We conclude that TSP-1 is involved in influencing the critical balance between MMPs and their inhibitors, maintaining the controlled degradation of the extracellular matrix needed to support metastasis and our results may provide an explanation for the divergent activities reported for TSP-1 in tumor progression.

Clinical significance of serum angiocidin levels in hepatocellular carcinoma.

Angiocidin, a tumor-secreted protein, was measured in serum of 27 healthy volunteers and 33 hepatocellular carcinoma (HCC) patients. Healthy controls either hepatitis B surface antigen (HBsAg) positive or negative showed undetectable levels. Patients had levels of angiocidin ranging from 15.09 to 195.73 pg/ml. Patients with stages III-IV had higher levels of angiocidin (97+/-13 pg/ml, n=17) compared to those with stages I-II (63+/-37 pg/ml, n=16), p<0.043. Patients with microsatellite tumor nodules had higher average levels (98+/-55 pg/ml, n=17) compared to those without microsatellite nodules (51+/-27 pg/ml, n=20), p<0.032. Our studies suggest that angiocidin predicts advanced stage and intra-hepatic metastasis.

Thrombospondin-1 (TSP-1) Stimulates Expression of Integrin alpha6 in Human Breast Carcinoma Cells: A Downstream Modulator of TSP-1-Induced Cellular Adhesion.

Thrombospondin-1 (TSP-1) is involved in a variety of different cellular processes including cell adhesion, tumor progression, and angiogenesis. This paper reports the novel finding that TSP-1 upregulates integrin alpha6 subunit in human keratinocytes and human breast cancer cells resulting in increased cell adhesion and tumor cell invasion. The effect of TSP-1 on alpha6 subunit expression was examined in human keratinocytes and breast adenocarcinoma cell lines (MDA-MB-231) treated with TSP-1 and in TSP-1 stably transfected breast cancer cells. TSP-1 upregulated alpha6 message and protein in these cells as revealed by differential display, Northern and Western blot analysis and immunohistochemical localization studies. The increased expression of alpha6 was shown to mediate adhesion and invasion of these cells to laminin, a major component of the basement membrane and extracellular matrix (ECM). These data suggest that TSP-1 plays an integral role in the attachment of cells to the ECM facilitating cell motility and angiogenesis.

Effect of VP12 and viperistatin on inhibition of collagen-receptor-dependent melanoma metastasis.

Viperistatin and VP12 isolated from Vipera paleastinae venom showed a potent inhibitory activity against collagen receptors, alpha1beta1 and alpha2beta1 integrins, respectively. Structurally, viperistatin belongs to the disintegrin family of proteins, whereas VP12 is composed of two subunits VP12A and VP12B displaying amino acid sequence homology with heterodimeric C-lectin type proteins. Viperistatin and VP12 used separately and simultaneously inhibited pro-metastatic activities of melanoma cells lines. The level of inhibition of MV3 and HS.939T human cell lines in cell adhesion and migration assays by both compounds was correlated with expression of alpha1beta1 and alpha2beta1 integrins on the cell surface. MV3 cells express collagen receptors to much higher extent than HS.939T and required the application of higher concentrations of inhibitors to block their adhesion to collagen types I and IV. A melanoma cell transmigration assay through a dHMVEC layer revealed that alpha1beta1 integrin plays a significant role in invasion of HS.939T cells, while alpha2beta1 integrin appears to be more important for MV3 cells. In an animal model of hematogenous metastasis of the mouse B16F10 cell line, the inhibitory effect of viperistatin and VP12 was only partial. These data suggest that collagen receptors may be an interesting target for development of new anti-metastatic therapies.

The novel angiogenic inhibitor, angiocidin, induces differentiation of monocytes to macrophages.

We previously showed that angiocidin, a tumor and vascular associated protein, is a potent inhibitor of angiogenesis and tumor growth. Angiocidin is a multidomain protein that exerts its antiangiogenic activity through multiple mechanisms, including effects on cell matrix interaction. Here, we describe another activity of angiocidin that may contribute to its antitumor activity. We show that angiocidin activates monocytes to secrete a mixture of proinflammatory cytokines and induces them to differentiate into macrophage-like cells. Using the monocytic cell line THP-1, we show that angiocidin induces the cells to become adherent and phagocytic, express macrophage markers, and secrete matrix metalloproteinase-9. Microarray analysis of control and angiocidin-treated THP-1 cells revealed that angiocidin up-regulated p105/p50, p100/p52, and rel B, components of the nuclear factor-kappaB (NF-kappaB) pathway. We confirmed the microarray data and showed that angiocidin induced phosphorylation of I kappa beta, p50, and p65 and translocation of p50 and p65 to the nucleus. We also showed that angiocidin activated up-stream mediators of NF-kappaB, such as the mitogen-activated protein kinase (MAPK) pathway and phosphoinositide-3 kinase (PI3K). Blockage of NF-kappaB and MAPK activation with small molecule inhibitors completely prevented angiocidin-mediated secretion of cytokines from THP-1 cells, but did not inhibit their adhesive phenotype. Blocking PI3K inhibited both secretion of cytokines, as well as the adhesive phenotype. These data suggest that angiocidin activates monocytes to secrete cytokines and differentiates them to a macrophage-like phenotype through at least two pathways mediated by MAPK and NF-kappaB, as well as PI3K.

Reduction of angiocidin expression in human umbilical vein endothelial cells via siRNA silencing inhibits angiogenesis.

Angiocidin, a protein over-expressed in many different solid tumors and tumor capillary endothelial cells inhibits angiogenesis and tumor growth [Zhou, J., et al., 2004. Cloning and characterization of angiocidin, a tumor cell binding protein for thrombospondin-1. J Cell Biochem. 92, 125-146]. Since several splice variants of angiocidin have distinct biochemical functions in membrane transport and protein degradation, we sought to evaluate the function of endogenously expressed angiocidin in human umbilical vein endothelial (HUVE) cells using siRNA. We observed a 90% reduction of the target mRNA levels after 24 h. Endogenous angiocidin protein expression was reduced by 80% after three days, as evaluated by Western blot analysis. We also found that anti-angiocidin siRNA down-regulated 90% of the protein expression of matrix metalloproteinase 2 (MMP-2) and 50% of its gelatinolytic activity. Reduction of endogenous angiocidin completely inhibited endothelial cord formation on Matrigel. Cells expressing low levels of angiocidin grew more slowly, were less invasive and less adhesive than control cells. Consistent with the reported function of one of the angiocidin analogues S5a, we found that the expression of polyubiquitinated proteins was higher in anti-angiocidin siRNA-treated cells as compared to normal and control siRNA-treated cells. These results suggest that endogenous angiocidin and its homologues promote endothelial cell invasion, adhesion, and angiogenesis through mechanisms involving polyubiquitin-dependent protein degradation and MMP-2 expression.

Integrin alpha2beta1 mediates the anti-angiogenic and anti-tumor activities of angiocidin, a novel tumor-associated protein.

We recently characterized an anti-tumor protein termed angiocidin. Here, we report that angiocidin may inhibit angiogenesis by binding collagen and its receptors. Angiocidin bound purified type I collagen and alpha2beta1 with high affinity. K562 cells expressing alpha2beta1 bound and adhered to angiocidin while K562 cells which only expressed alpha5beta1 integrin showed no binding and adhesion. Binding was specific since a neutralizing antibody against alpha2beta1 inhibited binding but antibodies against alpha5beta1 had no effect. Additionally, angiocidin co-localized with alpha2beta1 on K562 alpha2beta1 transfected cells, pancreatic cancer colo 357 cells, breast cancer MB-231 cells and human umbilical endothelial vein (HUVE) cells. In an alpha2beta1-dependent collagen gel angiogenesis assay, angiocidin showed potent inhibitory activity. We identified a 20-amino-acid amino terminal peptide of angiocidin that bound both alpha2beta1 and type I collagen. This peptide promoted alpha2beta1-dependent cell adhesion and inhibited tumor growth and angiogenesis. Taken together, these results are consistent with the conclusion that the anti-tumor activity of angiocidin arises from its ability to ligate collagen and alpha2beta1 on endothelial cells and tumor cells. Our results provide support for the concept that targeting matrix-cell interactions is a viable strategy for the development of anti-cancer therapeutics.

Angiostatin binds to tyrosine kinase substrate annexin II through the lysine-binding domain in endothelial cells.

Angiostatin(AS), an internal fragment of plasminogen, is one of the most potent specific inhibitors of angiogenesis. Angiostatin treatment has resulted in the complete regression of human tumors implanted subcutaneously into nude mice and has great therapeutic value (O'Reilly et al., Nat. Med. 2, 689-692, 1996). Despite promising therapeutic value in the treatment of cancer, the mechanism of its action is still unknown. We found that angiostatin binds to a 35-kDa protein in bovine aortic endothelial (BAE) cells (Sharma et al., Proc. Am. Assoc. Cancer Res. 42, 568, A3050, 2002). In an attempt to begin to understand angiostatin's mechanism of action, we have purified and characterized this 35-kDa protein from BAE cells. Internal peptide sequence analysis of purified protein demonstrated (SLYYIQQDTK, SYSPYDMLESIK, and ALLYLXGGDD) 100% sequence identity with tyrosine kinase substrate annexin II. Solid phase binding analysis suggests that angiostatin specifically bound to purified annexin II immobilized on 96-well plastic plates. Hundred-fold molar excess of unlabeled AS and anti-annexin II antibody inhibited bindings 85 and 55%, respectively, suggesting specific interaction. Annexin II is a predominant receptor for angiostatin, since neutralizing the angiostatin by soluble receptor (annexin II) effectively blocks angiostatin's anti-EC activity. Similarly, saturating the annexin II receptor by plasminogen in endothelial cells also blocks angiostatin's activity. Both angiostatin and plasminogen bind to purified annexin II in BAE cells saturably with apparent K(d) values of 101 and 164 nM, respectively, for purified annexin II and K(d) values of 83 and 125 nM, respectively, for BAE cells. Anti-annexin II monoclonal antibody inhibited angiostatin and plasminogen binding to endothelial cells by 68 and 62%, respectively, supporting our in vitro studies that annexin II is a receptor for angiostatin. Angiostatin-binding protein/annexin II specifically expressed in endothelial cells but not in fibroblasts suggests its EC-specific function. Epsilon-aminocaproic acid, a lys analogue, effectively blocks angiostatin and annexin II interaction, indicating that the lysine-binding domain of AS is required for binding to annexin II. These results suggest that the antiangiogenic action of angiostatin may be mediated via interaction with annexin II. Identification of annexin II as a receptor for angiostatin provides further evidence that clotting and fibrinolytic pathways are directly involved in the angiogenic process.

Thrombospondin-1 promotes proliferative healing through stabilization of PDGF.

PURPOSE: Thrombospondin-1 (TSP-1) mediates chemotaxis, cell proliferation, angiogenesis, and protease regulation in healing. TSP-1 also binds platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-beta). This study confirms the role of TSP-1 and defines the relationship between TSP-1 and PDGF in proliferative tissue repair. METHODS: Purified TSP-1 was analyzed for bound PDGF. Cultured fibroblast growth response to TSP-1 and recombinant PDGF was studied and the effects of antibodies against TSP-1, PDGF, and TGF-beta on this response were evaluated. Levels of TSP-1 and PDGF and relative proteolytic activity in fluid collected from 10 skin graft donor sites were then assessed by ELISA and a protease assay kit. The effect of proteolysis on TSP-bound PDGF and free recombinant PDGF was studied by adding trypsin and measuring the remaining PDGF by ELISA. RESULTS: TSP-1 promoted dose-dependent fibroblast growth. While antibody to TGF-beta had no effect on promotion, antibody to both TSP-1 and PDGF eliminated this. Since a strong correlation of TSP-1 with PDGF levels was found and strong proteolysis was seen in all samples, we proposed that TSP-1 protected PDGF from proteolysis. Consistent with this, we found PDGF bound to TSP-1 was 33% less degraded than free PDGF upon trypsinization. CONCLUSIONS: These results suggest that TSP-1 stabilizes PDGF, enhancing the biological effects of PDGF in proliferative tissue repair. This effect of TSP-1 along with its matrix-modulating activities may have important clinical utility regarding topical growth factor therapy in wound healing, since high proteolytic activity is believed to be partially responsible for limiting the efficacy of this treatment.

Cloning and characterization of angiocidin, a tumor cell binding protein for thrombospondin-1.

Thrombospondin-1 (TSP-1) is a matrix protein that has been implicated in mechanisms of tumor progression. Our laboratory previously showed that the CSVTCG (cys-ser-val-thr-cys-gly) sequence of TSP-1 functioned as a tumor cell adhesion domain and CSVTCG peptides as well as an anti-peptide antibody possessed anti-metastatic activity in a murine model of lung metastasis. In a subsequent study, a putative TSP-1 binding protein from lung carcinoma was isolated by CSVTCG-peptide affinity chromatography. In this study, we present the full-length cDNA of this binding protein isolated from a prostate cancer cell (PC3-NI) cDNA library. The purified recombinant protein, termed angiocidin, is a potent inhibitor of tumor growth of Lewis Lung carcinoma in vivo and tumor invasion and angiogenesis in vitro. In addition, the recombinant protein inhibits tumor and endothelial cell proliferation and induces apoptosis. The activity of angiocidin both in vivo and in vitro is partially dependent on its TSP-1 binding activity, since an angiocidin deletion mutant missing a high affinity-binding site for TSP-1 failed to inhibit tumor growth in vivo and was less active in its anti-tumor and anti-angiogenic activities in vitro. These results suggest that the anti-tumor activity of TSP-1 reported in many studies may be mediated in part by binding proteins such as angiocidin. Such proteins may function as tumor-suppressor proteins, which limit the growth of tumors by inhibiting angiogenesis and cell matrix interaction.