George P. Tuszynski

The role of matrix metalloproteinases in tumor angiogenesis and tumor metastasis.

Although a considerable amount of effort has been placed on discovering the etiologies of cancer, the majority of the basic cancer research existing today has focused on understanding the molecular mechanism of tumor formation and metastasis. Metastatic spread of tumors continues to be a major obstacle to successful treatment of malignant tumors. Approximately 30% of those patients diagnosed with a solid tumor have a clinically detectable metastasis and for the remaining 70%, metastases are continually being formed throughout the life of the tumor. Even after the tumor is excised, the threat of death is attributable to the metastasis that may occur through the remaining tumor cells. In addition, treating the metastasis often proves futile since metastasis often vary in size, composition, and anatomical location. New treatments blocking the formation of metastasis will provide greater chances of survival for cancer patients. One family of enzymes that has been shown over the years to play a role in tumor progression is the matrix metalloproteinase (MMP) family. The main function of MMPs, also known as matrixins, is degradation of the extracellular matrix physiologic function involving MMPs include wound healing, bone resorption and mammary involution. MMPs, however, also contribute to pathological conditions including rheumatoid arthritis, coronary artery disease, and cancer. Tumor cells are believed to utilize the matrix degrading capability of these enzymes to spread to distant sites. In addition, MMPs also are thought to promote the growth of these tumor cells once they have metastasized. This review will discuss the role of MMPs and their inhibitors in tumor invasion, angiogenesis and metastasis with special emphasis on the gelatinases, MMP-2 and MMP-9.

A rapid method for removal of [125I]iodide following iodination of protein solutions

Abstract A simple, highly effective, and rapid method for the removal of [ 125 I]iodide following iodination of protein solutions is described. Greater than 98% of free iodide is removed in approximately 2 min with protein recoveries in excess of 90%. An advantage of the procedure is the confinement of radioactive waste to small easily disposable tubes.

The role of thrombospondin-1 in tumor progression.

The role of thrombospondin-1 (TSP-1) in tumor progression is both complex and controversial. It is clear from the literature that the function of TSP-1 in malignancy depends on the presence of other factors and the level of TSP-1 expression in the tumor tissue. High levels of TSP-1 secreted by tumors, which were engineered to overexpress TSP-1, inhibit tumor growth, while anti-sense inhibition of TSP-1 production in certain tumore also inhibits growth. Clearly, the presence of other factors in these experimental systems must be important. The role of TSP-1 in angiogenesis also depends on the levels of TSP-1, the presence and level of angiogenic stimulators such as basic fibroblast growth factor (bFGF), and the localization of TSP-1 in the tissue. Matrix-bound TSP-1 promotes capillary tube formation in the rat aorta model of angiogenesis, while TSP-1 inhibits bFGF- induced angiogenesis in the rat cornea model. The inhibitory effect also depends on the proteolytic state of TSP-1 since the amino terminus promotes angiogenesis in the cornea model, while the remaining140-kDa fragment inhibits bFGF-induced angiogenesis. Both the stimulatory and inhibitory effects of TSP-1 are likely due to upregulation of matrix-degrading enzymes and their inhibitors. These enzymes are critical for maintaining optimal matrix turnover during angiogenesis. These varied TSP-1-dependent mechanisms offer new targets for the development of anti-angiogenic therapeutics for the treatment of a variety of cancers, as well as other pathologies involving inappropriate angiogenesis such as diabetic retinopathy.

Interaction of α9β1 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 &agr;9&bgr;1 integrin, and has an integrin binding site within its N-terminal domain (NoC1). Interaction of thrombospondin-1 and its recombinant NoC1 domain with &agr;9&bgr;1 integrin was confirmed in ELISA and cell adhesion assays. Binding of NoC1 to cells expressing &agr;9&bgr;1 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 &agr;9&bgr;1 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 &agr;9&bgr;1 inhibitors. In summary, our results revealed that &agr;9&bgr;1 integrin expressed on microvascular endothelial cells interacts with thrombospondin-1, and this interaction is involved in modulation of angiogenesis.

Expression of Thrombospondin-1 in Cancer: A Role in Tumor Progression

Abstract Thrombospondin-1 (TSP-1), a trimeric high molecular weight glycoprotein, is one of the major secreted proteins of human platelets and an extracellular matrix component of a variety of cells including vascular endothelial cells and tumor cells. TSP-1 has been shown to be highly expressed in human malignant tissues and present in higher than normal levels in the plasma of cancer patients. TSP-1 has also been shown to promote hematogenous tumor spread, tumor cell adhesion and invasion, and angiogenesis. Overall these studies provide compelling evidence for the conclusion that TSP-1 plays an important role in tumor progression.

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.

Interaction of platelet factor 4 with human platelets.

Human washed resting platelets bound 125I-labeled platelet factor 4 in a reaction which was saturable and approached equilibrium within 15-30 min. Scatchard plot analysis of the binding isotherms suggested a single class of specific binding sites. Excess of unlabeled protein and low- and high-affinity heparin competed for platelet factor 4 binding sites on the platelet surface and caused a partial displacement of this molecule. Anti-platelet factor 4 Fab fragments caused inhibition of binding of 125I-platelet factor 4 to platelets. Most of the labeled platelet factor 4 which was bound to intact platelets was recovered in the Triton X-100-insoluble cytoskeletal fraction prepared from the same platelets after their stimulation by thrombin. The association with the cytoskeleton was inhibited by anti-platelet factor 4 Fab fragments and by low-affinity heparin. Anti-platelet factor 4 125I-labeled Fab fragments bound to resting platelets, and this binding was greatly increased following platelet stimulation with thrombin. This suggested that endogenously secreted platelet factor 4 also binds to the platelet surface. No significant binding to platelets of 125I-labeled beta-thromboglobulin and 125I-labeled anti-beta-thromboglobulin Fab fragments was observed. Fab fragments of monospecific anti-human platelet factor 4 antibody raised in rabbits inhibited platelet aggregation and secretion induced by low concentrations of thrombin. Fab fragments of anti-beta-thromboglobulin antibody had no inhibitory effect. We suggest that the binding of alpha-granule-derived platelet factor 4 to the specific sites on the surface of platelets may modulate platelet aggregation and secretion induced by low levels of platelet agonists.

Trigramin, an RGD-containing peptide from snake venom, inhibits cell-substratum adhesion of human melanoma cells☆

Trigramin, a cysteine-rich, RGD-containing peptide isolated from the venom of the Trimeresurus gramineus snake, inhibited the adhesion of human melanoma cells to fibronectin and fibrinogen. Compared on a molar basis to GRGDSP, trigramin was approximately 500 times more potent than the hexapeptide at inhibiting cell adhesion to fibronectin. The activity of trigramin was abolished by chemical reduction of the molecule, indicating that the secondary structure is important to the biological activity. Trigramin presents an example of an effective inhibitor of cell adhesion that has developed in nature and may prove to be a useful probe in studying the cell surface receptors involved in cell adhesion.

Thrombospondin-1 and transforming growth factor-betal promote breast tumor cell invasion through up-regulation of the plasminogen/plasmin system

BACKGROUND Pericellular proteolysis is crucial in tumor cell invasion. The plasminogen/plasmin system is one of the main protease systems involved in cancer progression. Thrombospondin-1 (TSP-1), through activation of transforming growth factor-beta 1 (TGF-beta 1), up-regulates the main plasminogen activator, the urokinase-type plasminogen activator (uPA). The objectives of this study were to determine the role of TSP-1 and TGF-beta 1 in the localization of the plasminogen/plasmin system to the tumor cell surface by the uPA receptor (uPAR) and to determine its effect in breast tumor cell invasion. METHODS The effect of TSP-1 and TGF-beta 1 in uPAR expression was determined in MDA-MB-231 human breast cancer cells by enzyme-linked immunosorbent assay and Western blot analysis. Their effect and the role of the plasminogen/plasmin system in breast tumor cell invasion were studied with a Boyden Chamber assay. RESULTS uPAR expression was up-regulated more than twofold by both TSP-1 and TGF-beta 1. The effect of TSP-1 involved its receptor and the activation of TGF-beta 1 by TSP-1. Breast tumor cell invasion was up-regulated sevenfold to eightfold by both TSP-1 and TGF-beta 1 compared with the control group. Antibodies against uPA or uPAR neutralized the TSP-1- and TGF-beta 1-promoted breast tumor cell invasion. CONCLUSIONS TSP-1, through the activation of endogenous TGF-beta 1, up-regulates the plasminogen/plasmin system and promotes tumor cell invasion in breast cancer cells.

Adhesive Proteins and the Hematogenous Spread of Cancer

The metastatic spread of cancer is a complex and multistep process characterized by a number of biological steps which include the hematogenous and lymphatic arrest and adhesion of circulating tumor cells in the vascular bed, invasion of tumor cells through the basement membrane, and growth of new tumor colonies in the organ parenchyma. In this brief review we describe the role of platelets, the hemostatic system, adhesive proteins and their putative receptors in the hematogenous dissemination of cancer. The major adhesive proteins postulated to play a role in tumor arrest in the vascular bed are thrombospondin-1 (TSP-1), laminin, fibronectin and hyaluronan-proteoglycans. The major tumor and vascular receptors mediating these adhesive interactions are the CSVTCG-specific TSP-1 receptor, the 67-kD laminin receptor, the alpha v beta 3 vitronectin/TSP-1/fibronectin receptor, and CD44 hyaluronan receptor. The discovery of the involvement of these adhesive proteins and receptors in the metastatic spread of cancer as well as components of the hemostatic system offers unique opportunities for the development of antimetastasis therapies for the treatment of cancer.