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Stanley Zucker

Publications and source records attributed to Stanley Zucker.

At least 19 recordsLinked to original sources
PubMed2006

Neutrophil activator of matrix metalloproteinase-2 (NAM).

We have isolated a novel soluble factor(s), neutrophil activator of matrix metalloproteinases (NAM), secreted by unstimulated normal human peripheral blood neutrophils that causes the activation of cell secreted promatrix metalloproteinase-2 (proMMP-2). Partially purified preparations of NAM have been isolated from the conditioned media of neutrophils employing gelatin-Sepharose chromatography and differential membrane filter centrifugation. NAM activity, as assessed by exposing primary human umbilical vein endothelial cells (HUVEC) or HT1080 cells to NAM followed by gelatin zymography, was seen within one hour. Tissue inhibitor of metalloproteinase-2 (TIMP-2) and hydroxamic acid derived inhibitors of MMPs (CT1746 and BB94) abrogated the activation of proMMP-2 by NAM, while inhibitors of serine and cysteine proteases showed no effect. NAM also produced an increase in TIMP-2 binding to HUVEC and HT1080 cell surfaces that was inhibited by TIMP-2, CT1746, and BB94. Time-dependent increases in MT1-MMP protein and mRNA were seen following the addition of NAM to cells. These data support a role for NAM in cancer dissemination.

Adult↗
PubMed2006

The protease complex consisting of dipeptidyl peptidase IV and seprase plays a role in the migration and invasion of human endothelial cells in collagenous matrices.

Dipeptidyl peptidase IV (DPP4/CD26) and seprase/fibroblast activation protein alpha are homologous type II transmembrane, homodimeric glycoproteins that exhibit unique prolyl peptidase activities. Human DPP4 is ubiquitously expressed in epithelial and endothelial cells and serves multiple functions in cleaving the penultimate positioned prolyl bonds at the NH(2) terminus of a variety of physiologically important peptides in the circulation. Recent studies showed a linkage between DPP4 and down-regulation of certain chemokines and mitogenic growth factors, and degradation of denatured collagens (gelatin), suggesting a role of DPP4 in the cell invasive phenotype. Here, we found the existence of a novel protease complex consisting of DPP4 and seprase in human endothelial cells that were activated to migrate and invade in the extracellular matrix in vitro. DPP4 and seprase were coexpressed with the three major protease systems (matrix metalloproteinase, plasminogen activator, and type II transmembrane serine protease) at the cell surface and organize as a complex at invadopodia-like protrusions. Both proteases were colocalized at the endothelial cells of capillaries, but not large blood vessels, in invasive breast ductal carcinoma in vivo. Importantly, monoclonal antibodies against the gelatin-binding domain of DPP4 blocked the local gelatin degradation by endothelial cells in the presence of the major metallo- and serine protease systems that modified pericellular collagenous matrices and subsequent cell migration and invasion. Thus, we have identified a novel mechanism involving the DPP4 gelatin-binding domain of the DPP4-seprase complex that facilitates the local degradation of the extracellular matrix and the invasion of the endothelial cells into collagenous matrices.

Antibodies, Monoclonal↗
PubMed2006

Loss of intercellular adhesion activates a transition from low- to high-grade human squamous cell carcinoma.

The relationship between loss of intercellular adhesion and the biologic properties of human squamous cell carcinoma is not well understood. We investigated how abrogation of E-cadherin-mediated adhesion influenced the behavior and phenotype of squamous cell carcinoma in 3D human tissues. Cell-cell adhesion was disrupted in early-stage epithelial tumor cells (HaCaT-II-4) through expression of a dominant-negative form of E-cadherin (H-2Kd-Ecad). Three-dimensional human tissue constructs harboring either H-2Kd-Ecad-expressing or control II-4 cells (pBabe, H-2Kd-EcadDeltaC25) were cultured at an air-liquid interface for 8 days and transplanted to nude mice; tumor phenotype was analyzed 2 days and 2 and 4 weeks later. H-2Kd-Ecad-expressing tumors demonstrated a switch to a high-grade aggressive tumor phenotype characterized by poorly differentiated tumor cells that infiltrated throughout the stroma. This high-grade carcinoma revealed elevated cell proliferation in a random pattern, loss of keratin 1 and diffuse deposition of laminin 5 gamma2 chain. When II-4 cell variants were seeded into type I collagen gels as an in vitro assay for cell migration, we found that only E-cadherin-deficient cells detached, migrated as single cells and expressed N-cadherin. Function-blocking studies demonstrated that this migration was matrix metalloproteinase-dependent, as GM-6001 and TIMP-2, but not TIMP-1, could block migration. Gene expression profiles revealed that E-cadherin-deficient II-4 cells demonstrated increased expression of proteases and cell-cell and cell-matrix proteins. These findings showed that loss of E-cadherin-mediated adhesion plays a causal role in the transition from low- to high-grade squamous cell carcinomas and that the absence of E-cadherin is an important prognostic marker in the progression of this disease.

Animals↗
PubMed2006

Detection of activated, TIMP-free MMPs.

MMP inhibitor-tethered resins provide a method for detection of activated MMPs in tissues. These measurements will help to elucidate in situ mechanisms of MMP activation, further our understanding of the tumor microenvironment, and facilitate development of MMP-inhibitor therapy.

Enzyme Activation↗
PubMed2006

Plasma matrix metalloproteinases 7 and 9 in patients with metastatic breast cancer treated with marimastat or placebo: Eastern Cooperative Oncology Group trial E2196.

BACKGROUND: The purpose of this study was to evaluate the prognostic and predictive utility of measuring plasma levels of matrix metalloproteinase (MMP)-7 and MMP-9 in patients with metastatic breast cancer (MBC) treated with the oral MMP inhibitor (MMPI) marimastat or a placebo. PATIENTS AND METHODS: We measured plasma levels of MMP-7 and MMP-9 using an enzyme-linked immunoassay method in 140 evaluable patients with MBC enrolled on a multicenter clinical trial that compared the MMPI marimastat with placebo. Specimens were collected after completion of first-line chemotherapy in patients who had responding or stable disease and 3, 6, and 12 months after initiation of marimastat (10 mg orally twice daily) or placebo. RESULTS: Baseline plasma MMP-7 and MMP-9 levels did not correlate with each other or with progression-free or overall survival. In addition, serial evaluation of plasma MMP-7 and MMP-9 levels revealed no significant changes over time in the marimastat or placebo groups or any correlation with trough plasma marimastat level. CONCLUSION: We conclude that measurement of plasma MMP-7 or MMP-9 levels, as performed in our trial, was not a useful prognostic or predictive factor in patients with MBC or in patients treated with an MMPI.

Administration, Oral↗
PubMed2005

Cyclic mechanical strain-induced proliferation and migration of human airway smooth muscle cells: role of EMMPRIN and MMPs.

Airway smooth muscle (ASM) proliferation and migration are major components of airway remodeling in asthma. Asthmatic airways are exposed to mechanical strain, which contributes to their remodeling. Matrix metalloproteinase (MMP) plays an important role in remodeling. In the present study, we examined if the mechanical strain of human ASM (HASM) cells contributes to their proliferation and migration and the role of MMPs in this process. HASM were exposed to mechanical strain using the FlexCell system. HASM cell proliferation, migration and MMP release, activation, and expression were assessed. Our results show that cyclic strain increased the proliferation and migration of HASM; cyclic strain increased release and activation of MMP-1, -2, and -3 and membrane type 1-MMP; MMP release was preceded by an increase in extracellular MMP inducer; Prinomastat [a MMP inhibitor (MMPI)] significantly decreased cyclic strain-induced proliferation and migration of HASM; and the strain-induced increase in the release of MMPs was accompanied by an increase in tenascin-C release. In conclusion, cyclic mechanical strain plays an important role in HASM cell proliferation and migration. This increase in proliferation and migration is through an increase in MMP release and activation. Pharmacological MMPIs should be considered in the pursuit of therapeutic options for airway remodeling in asthma.

Asthma↗
PubMed2005

Human carcinoma cell growth and invasiveness is impaired by the propeptide of the ubiquitous proprotein convertase furin.

Furin, a potent proprotein convertase involved in activation of several cancer-related substrates, is synthesized as an inactive zymogen, thus minimizing the occurrence of premature enzymatic activity that would lead to inappropriate protein activation or degradation. This natural inhibitory mechanism is based on the presence of an inactivating prosegment at the NH2 terminal of the zymogen. After initial autocatalytic cleavage, the prosegment remains tightly associated with the convertase until it reaches the trans-Golgi network where the dissociation of the prosegment and activation of furin occurs. We hypothesized that the inhibitory properties of the preprosegment of furin (ppFur) could be beneficial if ectopically expressed in tumor cells. Transfection of four human head and neck squamous cell carcinoma cell lines with the complete ppFur cDNA sequence (pIRES-EGFP-ppFur) or with the empty expression vector (pIRES-EGFP) was done. The inhibitory effect was evaluated using in vivo tumorigenicity, invasion, anchorage-independent growth in soft agar, and proliferation assays, as well as by investigating impairment of furin substrates processing. Following transfection of ppFur, a significant reduction in cell proliferation, tumorigenicity, and invasiveness was observed in vitro and in vivo. These biological changes are directly related to the inhibition of furin-mediated activation of crucial cancer-related substrates, such as membrane type 1 matrix metalloproteinase, transforming growth factor-beta, insulin-like growth factor-1 receptor, and vascular endothelial growth factor-C. PpFur expression in head and neck squamous cell carcinoma cell lines showed a mechanistic link between furin inhibition, decreased substrate processing, cell proliferation, and invasive ability. These findings suggest that furin inhibition is a feasible approach to ameliorate and even abolish the malignant phenotype of various malignancies.

Carcinoma, Squamous Cell↗
PubMed2005

Roles of the multifunctional glycoprotein, emmprin (basigin; CD147), in tumour progression.

Emmprin (basigin;CD147) is a widely distributed cell surface glycoprotein that belongs to the Ig superfamily and is highly enriched on the surface of malignant tumour cells. Emmprin is involved in numerous physiological and pathological systems and exhibits several molecular and cellular characteristics, but a major function of emmprin is stimulation of synthesis of several matrix metalloproteinases. In tumours, emmprin most likely stimulates matrix metalloproteinase production in stromal fibroblasts and endothelial cells as well as in tumour cells themselves by a mechanism involving homophilic interactions between emmprin molecules on apposing cells or on neighbouring cells after membrane vesicle shedding. Membrane-associated cofactors, including caveolin-1 and annexin II, regulate emmprin activity. Emmprin induces angiogenesis via stimulation of VEGF production, invasiveness via stimulation of matrix metalloproteinase production and multidrug resistance via hyaluronan-mediated up-regulation of ErbB2 signaling and cell survival pathway activities. Although the detailed mechanisms whereby it regulates these numerous phenomena are not yet known, it is clear that emmprin is a major mediator of malignant cell behavior.

Antigens, CD↗
PubMed2005

The organomercurial 4-aminophenylmercuric acetate, independent of matrix metalloproteinases, induces dose-dependent activation/inhibition of platelet aggregation.

Matrix metalloproteinases (MMPs) play an important role in many biological and pathological processes including tissue remodeling, wound healing, inflammation, atherosclerosis, and cancer. Numerous publications have supported the concept that activated MMP-2 enhances agonist-induced platelet aggregation and activated MMP-9 inhibits platelet aggregation. In this study, we demonstrated that the organomercurial compound, 4-aminophenyl mercuric acetate (APMA), which is routinely employed to activate latent MMPs at a concentration of 1000 microM, induces platelet aggregation at low concentration (5 microM) and inhibits agonist-induced platelet aggregation at concentrations >or= 50 microM. Activated MMP-2, MMP-1, and MMP-9, following removal of APMA by ultrafiltration through an anisotropic membrane, exert no independent effect on platelet aggregation. Acetylsalicylic acid and BAPTA inhibited APMA-induced platelet aggregation indicating that the APMA mediated pathway of platelet activation is dependent upon thromboxane and calcium signaling. Zinc chelation with 1,10-phenanthroline, which inhibits zinc-dependent proteins including metalloproteinases, also abrogated platelet functional responses to APMA. Additional studies will be required to clarify the mechanism of the biphasic effect of APMA on platelet aggregation.

Calcium Signaling↗
PubMed2004

Randomized phase III trial of marimastat versus placebo in patients with metastatic breast cancer who have responding or stable disease after first-line chemotherapy: Eastern Cooperative Oncology Group trial E2196.

PURPOSE: To determine whether a matrix metalloproteinase inhibitor improves progression-free survival (PFS) in patients with metastatic breast cancer who have responding or stable disease after first-line chemotherapy. PATIENTS AND METHODS: One hundred seventy-nine eligible patients were randomly assigned to receive oral marimastat (10 mg bid; n = 114) or a placebo (n = 65) within 3 to 6 weeks of completing six to eight cycles of first-line doxorubicin- and/or taxane-containing chemotherapy for metastatic disease. Patients were evaluated every 3 months until disease progression. RESULTS: When comparing placebo with marimastat, there was no significant difference in PFS (median, 3.1 months v 4.7 months, respectively; hazard ratio, 1.26; 95% CI, 0.91 to 1.74; P = .16) or overall survival (median, 26.6 months v 24.7 months, respectively; hazard ratio, 1.03; 95% CI, 0.73 to 1.46; P = .86). Patients treated with marimastat were more likely to develop grade 2 or 3 musculoskeletal toxicity (MST), a known complication of the drug indicative of achieving a biologic effect, compared with patients administered placebo (63% v 22%, respectively; P < .0001). Patients with grade 2 or 3 MST had significantly inferior survival compared with patients who had grade 0 or 1 MST (median, 22.5 months v 28.2 months; P = .04). In addition, patients who had a marimastat plasma concentration of at least 10 ng/mL at month 1 and/or 3 were significantly more likely to have grade 2 to 3 MST (P < .0001). CONCLUSION: Marimastat does not prolong PFS when used after first-line chemotherapy for metastatic breast cancer. Patients with higher marimastat levels exhibited MST, and MST was associated with inferior survival.

Administration, Oral↗
PubMed2004

Intracellular processing and activation of membrane type 1 matrix metalloprotease depends on its partitioning into lipid domains.

The integral membrane type 1 matrix metalloprotease (MT1-MMP) is a pivotal protease in a number of physiological and pathological processes and confers both non-tumorigenic and tumorigenic cell lines with a specific growth advantage in a three-dimensional matrix. Here we show that, in a melanoma cell line, the majority (80%) of MT1-MMP is sorted to detergent-resistant membrane fractions; however, it is only the detergent-soluble fraction (20%) of MT1-MMP that undergoes intracellular processing to the mature form. Also, this processed MT1-MMP is the sole form responsible for ECM degradation in vitro. Finally, furin-dependent processing of MT1-MMP is shown to occur intracellularly after exit from the Golgi apparatus and prior to its arrival at the plasma membrane. It is thus proposed that the association of MT1-MMP with different membrane subdomains might be crucial in the control of its different activities: for instance in cell migration and invasion and other less defined ones such as MT1-MMP-dependent signaling pathways.

Biotinylation↗
PubMed2004

CXCR4-mediated adhesion and MMP-9 secretion in head and neck squamous cell carcinoma.

The chemokine CXCL12 (SDF-1) and its receptor, CXCR4, have been implicated in organ-specific metastases of several malignancies. Head and neck squamous cell carcinoma (HNSCC) predominantly metastasizes to lymph nodes, and recent evidence has shown that CXCL12 stimulates HNSCC migration. We explored the potential role of CXCR4 in mediating other metastatic processes in HNSCC cells. CXCR4 mRNA and cell-surface expression was assessed in HNSCC cell lines. CXCR4 mRNA expression was detected in five HNSCC cell lines. Cell-surface CXCR4 was also detected in each of the HNSCC cell lines and in resected HNSCC tissues. CXCL12 induced rapid intracellular calcium mobilization in a metastatic HNSCC cell line (HN), as well as rapid phosphorylation of ERK-1/2. HNSCC cell adhesion to fibronectin and collagen was increased by CXCL12 treatment, while the addition of an inhibitor of ERK-1/2 signaling, PD98059, reduced the effects of CXCL12. CXCL12 also increased the active matrix metalloproteinase (MMP)-9 secreted. Thus, HNSCC cells express functional CXCR4 receptors that induce rapid intracellular signaling upon binding to CXCL12. Such binding leads to increased HNSCC cell adhesion and MMP secretion, suggesting that CXCR4 may be a novel regulator of HNSCC metastatic processes.

Blotting, Western↗
PubMed2004

CXCR4 and CXCL12 (SDF-1) in prostate cancer: inhibitory effects of human single chain Fv antibodies.

PURPOSE: Metastasis is a major cause of morbidity in prostate cancer (PCa). Several studies have shown that the chemokine receptor CXCR4 and its ligand, CXCL12 (stromal cell-derived factor-1), regulate tumor cell metastasis to specific organs. Recently, it was demonstrated that CXCL12 enhances PCa cell adhesion, migration, and invasion, implicating CXCR4 in PCa metastasis. In this study, we examined the inhibitory effects of anti-CXCR4 antibodies on CXCL12-mediated PCa cell activities. EXPERIMENTAL DESIGN: We developed fully human single chain Fv antibodies (scFv), Ab124 and Ab125, against CXCR4 using the yeast two-hybrid system. We performed immunofluorescent staining, flow cytometry, and ELISA-binding assays to measure scFv binding to PCa cells. We also examined the effects of scFv on CXCL12-mediated calcium mobilization, cell migration, and invasion. RESULTS: Our results confirmed that PCa cell lines express cell-surface CXCR4. Real-time quantitative reverse transcription-PCR and immunohistochemical staining also verified that CXCR4 is expressed in primary cultures of prostate epithelial cells from adenocarcinomas and in human prostate tissues. Ab124 and Ab125 demonstrated specific binding to PCa cell lines by flow cytometry and in binding assays. Preincubation with scFv resulted in significant reduction of CXCL12-induced calcium mobilization in PC-3 and LNCaP cells. Ab124 and Ab125 also inhibited PCa cell migration toward CXCL12, as well as invasion through extracellular matrix gels. CONCLUSIONS: Ab124 and Ab125 inhibit CXCL12-mediated cellular activities by binding the receptor CXCR4. Recombinant scFv are an efficient mode of targeting tumor antigens. Considering the high incidence of PCa, the development of fully human scFv may be a useful therapeutic approach in the prevention and treatment of PCa metastasis.

Breast Neoplasms↗
PubMed2004

Prostate cancer cell adhesion to bone marrow endothelium: the role of prostate-specific antigen.

Bone metastasis is the most frequent complication of prostate cancer (PC). Elucidation of the biological basis of this specificity is required for the development of approaches for metastatic inhibition. We investigated the possibility that the preferential attachment of PC cells to bone marrow endothelium (as opposed to endothelium from other organs) affects this specificity. We selected, from peptide phage-displayed libraries, peptide ligands to surfaces of PC cells (C4-2B) attenuated (30-40%) binding of C4-2B cells to bone marrow endothelial cells (BMECs). We then determined the molecules on the surface of C4-2B cells interacted with the selected peptides using column affinity chromatography and a cDNA expression phage-displayed library generated from C4-2B cells in T7 phage. We identified a phage from the cDNA library that specifically bound to one of the selected peptides-L11. This phage displayed the amino acid sequence homologous for the COOH-terminal portion of prostate-specific antigen (PSA). To examine the possible direct involvement of PSA in the interactions between PC and BMECs, we performed a cell-cell adhesion assay. Antibodies to PSA attenuated PC cells adhesion to BMECs. In addition, exogenous proteolytically active PSA modulated this adhesion. Finally, inactivation of mRNA coding PSA by a small interfering RNA (siRNA) diminished C4-2B cell adhesion to BMECs. These results indicate that PSA expressed as secreted and surface-associated molecules in C4-2B cells is involved in cell-cell interactions and/or digests components of bone marrow endothelium for preferential adhesion and penetration of PC cells. The suggested experimental approach is a promising strategy for identification of cell surface molecules involved in intercellular interactions.

Apoptosis↗
PubMed2004

Emmprin promotes anchorage-independent growth in human mammary carcinoma cells by stimulating hyaluronan production.

Emmprin (CD147; basigin) is a plasma membrane glycoprotein, enriched on the surface of many cancer cells, which induces matrix metalloproteinase synthesis via cell-cell interactions. Elevated emmprin production causes increased growth in vivo of human mammary carcinoma cells. In this study, we show that elevation of emmprin expression in less aggressive human carcinoma cells, which normally express low emmprin levels, induces the ability to grow under anchorage-independent conditions. We also found that elevated emmprin expression stimulates hyaluronan production and that the effect of emmprin on anchorage-independent growth is dependent on hyaluronan. Furthermore, emmprin stimulates cell survival pathway signaling in a hyaluronan-dependent manner. From these and other studies we conclude that emmprin enhances several malignant properties of cancer cells, including anchorage-independent growth, invasiveness, and chemoresistance.

Antigens, CD↗
PubMed2004

TIMP-2 is released as an intact molecule following binding to MT1-MMP on the cell surface.

Binding of tissue inhibitor of metalloproteinase-2 (TIMP-2) to pro-MMP-2 and mature membrane type-1 MMP (MT1-MMP) on the cell surface is required for activation of MMP-2. It has been reported that following binding to cell surface receptors, TIMP-2 undergoes endocytosis and extensive degradation in lysosomes. The purpose of this study was to reexamine the fate of TIMP-2 following binding to transfected HT1080 cell surface MT1-MMP at 4 degrees C. Following 37 degrees C incubation, 125I-TIMP-2 release, endocytosis, and degradation were characterized under varying conditions. More than 85% of the total 125I-TIMP-2 bound to cells was released as intact functional molecules; <15% was degraded. Transfection of HT1080 cells with dominant negative mutant dynamin cDNA resulted in delayed endocytosis and release of 125I-TIMP-2 from cells. Pharmacologic agents that induce clustering of cell surface receptors (concanavalin A) and interfere with endosomal/lysosomal function (bafilomycin A(1)) resulted in enhanced binding of 125I-TIMP-2 to cell surface receptors. Abrogation of activation of proMT1-MMP with a furin inhibitor prevented binding and endocytosis of 125I-TIMP-2. Biotinylation of cell surface MT1-MMP followed by Western blotting confirmed the presence of mature MT1-MMP on the cell surface and degraded MT1-MMP in the intracellular compartment. In conclusion, these studies demonstrate that TIMP-2 is released from cells primarily as an intact functional molecule following binding to MT1-MMP on the cell surface.

Animals↗
PubMed2004

Distinct roles for the catalytic and hemopexin domains of membrane type 1-matrix metalloproteinase in substrate degradation and cell migration.

Substrate degradation and cell migration are key steps in cancer metastasis. Membrane-type 1-matrix metalloproteinase (MT1-MMP) has been linked with these processes. Using the fluorescein isothiocyanate (FITC)-labeled fibronectin degradation assay combined with the phagokinetic cell migration assay, structure-function relationships of MT1-MMP were studied. Our data indicate that MT1-MMP initiates substrate degradation and enhances cell migration; cell migration occurs as a concurrent but independent event. Using recombinant DNA approaches, we demonstrated that the hemopexin-like domain and a nonenzymatic component of the catalytic domain of MT1-MMP are essential for MT1-MMP-mediated cell migration. Because the cytoplasmic domain of MT1-MMP was not required for MT1-MMP-mediated fibronectin degradation and cell migration, it is proposed that cross-talk between the hemopexin domain of MT1-MMP and adjacent cell surface molecules is responsible for outside-in signaling. Employing cDNAs encoding dominant negative mutations, we demonstrated that Rac1 participates in the MT1-MMP signal transduction pathway. These data demonstrated that each domain of MT1-MMP plays a distinct role in substrate degradation and cell migration.

Animals↗