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Biomedical subjects

George P Tuszynski

Publications and source records attributed to George P Tuszynski.

15 recordsLinked to original sources
PubMed2006

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.

Blotting, Western↗
PubMed2006

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.

Amino Acids↗
PubMed2006

Antibody-directed targeting of angiostatin's receptor annexin II inhibits Lewis Lung Carcinoma tumor growth via blocking of plasminogen activation: possible biochemical mechanism of angiostatin's action.

Angiostatin, the N-terminal four kringles (K1-4) of parent molecule plasminogen, is reported to block Lewis Lung Carcinoma (LLC) tumor growth and metastasis. However, angiostatin's mechanism of action is unclear. We earlier reported that angiostatin binds to cell surface annexin II through the lysine-binding domain (kringles 1-4) [Tuszynski, G.P., Sharma, M., Rothman, V.L., Sharma, M.C., 2002. Angiostatin binds to tyrosine kinase substrate annexin II through the lysine-binding domain in endothelial cells. Microvasc. Res. 64:448-462.]). We now show that annexin II on the cell surface of LLC cells regulates conversion of plasminogen to plasmin. Activation of plasminogen to plasmin is time-dependent, with the linear activation lasting up to 120 min. Monoclonal antibodies to annexin II reduced plasminogen activation by 92.6%, suggesting a specific role of annexin II in plasmin generation. Angiostatin also reduced plasmin generation by 81.6%, suggesting that angiostatin may be competing with plasminogen through lysine-binding domain. epsilon-Aminocaproic acid, a lysine analogue, effectively blocked plasminogen activation indicating that, indeed, the lysine-binding site of the kringles domain is required for activation. These data suggest that annexin II may be a receptor target for angiostatin's action. Therefore, we tested the effect of high affinity monoclonal antibody to annexin II in mouse model of LLC. A single dose of antibody treatment inhibited LLC tumor growth almost 70% with concomitant inhibition of circulating plasmin generation and its proteolytic activity. Taken together, it is possible that inhibition of LLC tumor growth and metastasis reported by angiostatin therapy may be due to blocking of annexin-II-dependent plasmin generation. Plasmin is known to influence angiogenic, invasive and metastatic capability of tumors.

Angiogenesis Inhibitors↗
PubMed2006

Angiogenesis-associated protein annexin II in breast cancer: selective expression in invasive breast cancer and contribution to tumor invasion and progression.

Many advanced human tumors including breast cancer overproduce plasmin that is known to promote angiogenesis and metastasis. The mechanism of this effect is poorly understood. Here we report that annexin II, an endothelial co-receptor for tPA (tissue-type plasminogen activator) and plasminogen, was undetectable in normal and hyperplastic ductal epithelial cells and ductal complexes. By contrast, it was consistently expressed in invasive breast cancer and ductal carcinoma in situ (DCIS) indicating its involvement in breast cancer. Using the well established invasive/metastatic MDA-MB231 cell line and the noninvasive/nonmetastatic MCF-7 human breast cancer cell line, we investigated the mechanism by which annexin II regulates breast cancer progression and metastasis. Western and Northern blot analyses demonstrate selective expression of annexin II in MDA-MB231 cells but not in poorly invasive MCF-7 cells suggesting its participation in invasive breast cancer. Since annexin II is a receptor for plasminogen, we tested whether MDA-MB231 cells are capable of producing plasmin in vitro. MDA-MB231 cell membranes induced plasmin generation in a time-dependent manner while those from MCF-7 cells failed to convert plasminogen to plasmin. The generated plasmin is capable of degrading ECM consequently facilitating cell invasion and migration, biological functions required for angiogenesis and metastasis. Plasmin generation and its dependent invasion and migration can be blocked by a monoclonal antibody to annexin II or angiostatin, potent inhibitors of angiogenesis, breast cancer, and metastasis. Our findings indicate that annexin II-dependent localized plasmin generation by human breast cancer cells could contribute to angiogenesis and metastasis. These results suggest that annexin II may be an attractive target for new anti-angiogenic and anti-breast cancer therapies.

Angiostatins↗
PubMed2004

Clinical significance of thrombospondin 1 expression in hepatocellular carcinoma.

PURPOSE: Thrombospondin 1 (THBS 1) is a matricellular protein capable of modulating angiogenesis. However, the actual role of THBS 1 in angiogenesis and tumor progression remains controversial. Hepatocellular carcinoma (HCC) is a hypervascular tumor characterized by neovascularization. The significance of THBS 1 in HCC remains unknown. In this study, the significance of THBS 1 in HCC was evaluated by correlating its expression with clinicopathological data. The possible role of THBS 1 in the angiogenesis of HCC was also studied by correlating its expression with vascular endothelial growth factor (VEGF) expression. EXPERIMENTAL DESIGN: Sixty HCC patients were recruited in this study. THBS 1 and VEGF protein expression in tumorous livers were localized by immunohistochemical staining and quantified by ELISA. THBS 1 mRNA was quantified by quantitative reverse transcription-PCR. RESULTS: Immunohistochemical staining of THBS 1 was positive in HCC cells in 51.7% of patients and in stromal cells in 65% of patients. Tumor THBS 1 protein level was significantly correlated with its mRNA expression (P = 0.001) and was significantly correlated with tumor VEGF protein levels (P = 0.001). Its expression was significantly associated with the presence of venous invasion (P = 0.008) and advanced tumor stage (P = 0.049). High THBS 1 expression was also a prognostic marker of poor survival in HCC patients. CONCLUSIONS: This study shows that high expression of THBS 1 is associated with tumor invasiveness and progression in HCC. THBS 1 appears to be a proangiogenic factor that stimulates angiogenesis in HCC in view of its positive correlation with VEGF expression.

Aged↗
PubMed2004

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.

Amino Acid Sequence↗
PubMed2004

Angiostatin-induced inhibition of endothelial cell proliferation/apoptosis is associated with the down-regulation of cell cycle regulatory protein cdk5.

Endothelial cells (ECs) are quiescent in normal blood vessels, but undergo rapid bursts of proliferation after vascular injury, hypoxia or induced by powerful angiogenic cytokines like fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). Deregulated proliferation of ECs facilitates angiogenic processes and promotes tumor growth. In dividing cells, cell cycle-associated protein kinases, which are referred as cyclin-dependent kinases (cdks), regulate proliferation, differentiation, senescence, and apoptosis. Cyclin-dependent kinase-5 (cdk5) is expressed in neuronal cells and plays an important role in neurite outgrowth, of neuronal migration and neurogenesis, its functions in non-neuronal cells are unclear. Here, we show for the first time that the cdk5 is expressed at high levels in proliferating bovine aortic endothelial (BAE) cells, by contrast insignificant low levels of cdk5 expression in quiescent BAE cells. In addition, bFGF up-regulates cdk5 expression in a dose-dependent fashion. Interestingly, temporal expression data suggests that cdk5 expression is very low between 24-48 h, but high level of cdk5 expression was detected during 60-72 h. This later time corresponds to the time of completion of one cell cycle (doubling of cell population) of BAE cell culture. Angiostatin (AS), a powerful inhibitor of angiogenesis inhibits ECs proliferation in dose-dependent manner with concomitant down-regulation of cdk5 expression. The role of cdk5 in ECs, proliferation and apoptosis was confirmed by selective inhibition of cdk5 expression by the purine derivative roscovitine, which inhibits bFGF-stimulated BAE cells proliferation and induces apoptosis in dose-specific manner. By contrast, the roscovitine analog olomoucine, which is a specific inhibitor of cdk4, but not of cdk5 failed to affect ECs proliferation and apoptosis. These data suggest for the first time that neuron specific protein cdk5 may have significant role in the regulation of ECs proliferation, apoptosis, and angiogenesis and extends beyond its role in neurogenesis.

Angiogenesis Inhibitors↗
PubMed2004

Mechanisms of thrombospondin-1-mediated metastasis and angiogenesis.

The major route of tumor spread is through the bloodstream. Once in circulation, the tumor cells aggregate in clumps with platelets, which enhances the tumor cell survival. The tumor emboli will then adhere to the endothelium and by the release of proteases extravasation of the cells will occur. One of the platelet-secreted proteins is thrombospondin-1. In this article, thrombospondin-1 will be described as a modulator of angiogenesis through its role in regulating endothelial cell apoptosis, protease expression, and vascular endothelial growth factor expression. We hope to convey the idea that activity of thrombospondin-1 in tumor progression is dependent upon its interaction with several host- and tumor-associated proteins.

Endothelium, Vascular↗
PubMed2004

Antiangiogenic therapy.

Angiogenesis, the formation of blood vessels from preexisting ones, plays a crucial role in tumor progression. Activation of an "angiogenic switch" allows tumor cells to invade and metastasize. The growing interest in the use of antiangiogenic agents in the treatment and prevention of cancer lies in the theoretical advantages of this molecularly targeted modality of chemotherapy. Delivery of antiangiogenic agents are not complicated by having to penetrate large bulky masses but, instead, have easy access to tumoral endothelial cells. Antiangiogenic drugs may not cause cytopenias and thus will avoid many of the unwarranted toxicities of standard chemotherapeutic agents. Because they act directly on nascent endothelial cells, antiangiogenic agents may avoid tumor resistance mechanisms. If antiangiogenic agents are successful, they might be applicable to many tumor types and not be dependent on cell type or growth fraction of cells within a tumor. However, several important obstacles remain with regards to using antiangiogenic drugs in clinical trials with which we must contend in order to determine accurately the efficacy of these agents. In this article, we review the different classes of antiangiogenic agents available, ongoing clinical trials, as well as potential pitfalls and future directions in this exciting field.

Angiogenesis Inhibitors↗
PubMed2003

Obtustatin: a potent selective inhibitor of alpha1beta1 integrin in vitro and angiogenesis in vivo.

A novel disintegrin, obtustatin, was purified from the venom of the Vipera lebetina obtusa viper. Obtustatin is the shortest disintegrin yet described, containing only 41 amino acids. It contains a similar pattern of cysteines to the short disintegrins echistatin and eristostatin but contains the sequence KTS rather than RGD in its active site loop. Obtustatin is a potent and selective inhibitor of alpha1beta1 integrin. It does not inhibit the closely related integrin alpha2beta1, nor a panel of other integrins tested. It does not inhibit ligand binding to the recombinant alpha1 I-domain. Importantly, obtustatin potently inhibited angiogenesis in vivo in the chicken chorioallantoic membrane assay, and in the Lewis lung syngeneic mouse model, it reduced tumor development by half, confirming and extending previous results on the relevance of alpha1beta1 integrin to angiogenesis and suggesting novel approaches to the generation of angiogenesis inhibitors.

Allantois↗
PubMed2002

Up-regulation of matrix metalloproteinase 9 by thrombospondin 1 in gastric cancer.

BACKGROUND: Matrix metalloproteinase 9 (MMP-9) plays a key role in tumor cell invasion. It was recently reported that plasma levels of MMP-9 in patients with gastric cancer correlate with the tumors' metastatic potential. We previously demonstrated that thrombospondin 1 (TSP-1) up-regulates MMP-9 expression by endothelial cells and promotes tumor cell invasion. We hypothesized that TSP-1 plays a role in the up-regulation of MMP-9 in gastric cancer. METHODS: MMP-9, TSP-1, and CSVTCG-specific TSP-1 receptor expression were measured by immunohistochemical staining in 31 consecutive gastric adenocarcinomas from patients who did not undergo neoadjuvant chemotherapy or radiation therapy. Additionally, we measured TSP-1, CSVTCG-specific TSP-1 receptor, and MMP-9 expression by Western blotting, zymography, and immunohistochemical staining in AGS gastric adenocarcinoma cells. We also investigated the effect of TSP-1 on MMP-9 expression by AGS cells. RESULTS: TSP-1 localized to the tumor-associated extracellular matrix. CSVTCG-specific TSP-1 receptor and MMP-9 colocalized to tumor cells, fibroblasts, and tumor-associated microvessels. Intense staining for TSP-1, CSVTCG-specific TSP-1 receptor, and MMP-9 correlated with markers of aggressive tumor behavior. AGS gastric adenocarcinoma cells expressed high levels of CSVTCG-specific TSP-1 receptor but not TSP-1. TSP-1 up-regulated MMP-9 expression by AGS cells. CONCLUSIONS: We conclude that TSP-1 plays a role in the up-regulation of MMP-9 expression in gastric cancer. Our data also suggest a correlation between expression of TSP-1, CSVTCG-specific TSP-1 receptor, and MMP-9 and the acquisition of an aggressive tumor phenotype.

Adenocarcinoma↗
PubMed2002

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.

Cell Division↗
PubMed2002

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.

Angiostatins↗
PubMed2002

Fibroblasts promote breast cancer cell invasion by upregulating tumor matrix metalloproteinase-9 production.

BACKGROUND: Thrombospondin-1 (TSP-1) promotes breast cancer cell invasion of collagen by upregulating matrix metalloproteinase-9 (MMP-9) production. Stromal TSP-1 may play a role in regulating tumor cell invasion. We hypothesize that fibroblasts promote breast cancer cell invasion by upregulating the production of MMP-9 through TSP-1. METHODS: MDA-MB-231 human breast carcinoma cells were grown alone or in coculture with human fibroblasts. Gelatin zymography and Western immunoblot analysis for MMP-9 were performed on the coculture cell media and the single cell media. Inhibition of fibroblast-mediated breast tumor cell invasion by an anti-TSP-1 or an anti-MMP-9 antibody was evaluated using a modified Boyden chamber. RESULTS: Coculture experiments showed an increased production of MMP-9 when compared with breast cancer single cell culture or fibroblast single cell culture experiments as demonstrated by zymography and Western immunoblot analysis. Fibroblast-stimulated MMP-9 production was comparable with TSP-1-stimulated MMP-9 production. Anti-TSP-1 antibody and anti-MMP-9 antibody inhibited fibroblast-stimulated tumor cell invasion to 30% and 26% of controls, respectively (P <.05). CONCLUSIONS: Fibroblasts may regulate breast cancer cell invasion by promoting tumor MMP-9 production through TSP-1. Inhibition of stromal TSP-1 stimulation of MMP-9 synthesis may prevent matrix degradation necessary for tumor invasion and metastasis.

Antibodies↗