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V Vetvicka

Publications and source records attributed to V Vetvicka.

At least 19 recordsLinked to original sources
PubMed2005

Possible role of procathepsin D in human cancer.

For the past ten years, our research has been focused on elucidating the mechanism by which procathepsin D (pCD) impacts cancer development. Various studies have shown that pCD is overexpressed and secreted by numerous cancer cell lines. After secretion, it exhibits "growth hormone-like" activity on cancerous cells but the exact mechanism of this mitogenic activity is not yet understood. The activation peptide of pCD (APpCD) (which is cleaved off upon activation of the zymogen) is responsible for the mitogenic function of pCD. Various in vitro and in vivo studies support our theory that the APpCD interacts with both parent and neighborhood cancer cells and thus functions as an autocrine mitogen. We propose a model of pCD mitogenic function and also some possible approaches for treatment and prevention of certain types of cancer.

Biomarkers, Tumor↗
PubMed2002

Detection of procathepsin D in rat milk.

The presence of procathepsin D, a zymogen of the soluble lysosomal aspartic proteinase cathepsin D, was detected in rat milk using Western blot analysis and assay of proteolytic activity in acidic buffers. No other forms of cathepsin D were found. Two different polyclonal anti-procathepsin D antibodies were used for immunochemical detection of procathepsin D. Both antibodies we found to recognize rat procathepsin D. Proteolytic activity in acidic buffers was detected using a fluorogenic substrate specific for cathepsin D and was abolished by pepstatin A, a specific inhibitor of aspartic proteinases. This study represents third demonstration of presence of procathepsin D in mammal breast milk. Potential sources and physiological functions are discussed.

Amino Acid Sequence↗
PubMed2000

Role of procathepsin D activation peptide in prostate cancer growth.

BACKGROUND: Enzymatically inactive procathepsin D secreted from cancer cells has been confirmed to play a role in breast cancer development. We focused on prostate cancer and the role of activation peptide in mitogenic activity. METHODS: Synthetic peptides and monoclonal antibodies raised against individual fragments of activation peptide were employed. Cell proliferation was measured by MTT (3-[4,5-dimethylthiatol-2-yl]-2,5-diphenyl tetrazolium bromide) assay or by in vivo growth in nude mice. RESULTS: We demonstrated that the growth factor activity of activation peptide is localized in amino-acid region 27-44. In addition, both anti-activation peptide and anti-27-44 peptide antibodies administered in vivo inhibited the growth of human prostate tumors in mice. CONCLUSIONS: Based on these data, we hypothesize that the interaction of procathepsin D activation peptide with an unknown receptor is mediated by amino-acid sequence 27-44. This interaction leads in certain types of tumor to a proliferation and higher motility. Blocking of this interaction by antibodies or antagonists might be a valuable tool in prostate cancer inhibition.

Animals↗
PubMed2000

Critical role of Kupffer cell CR3 (CD11b/CD18) in the clearance of IgM-opsonized erythrocytes or soluble beta-glucan.

Liver macrophages (Kupffer cells) play a major role in blood clearance of both C3-opsonized immune complexes and therapeutic beta-glucan polysaccharides. Human Kupffer cells express three types of C3-receptors: CR1 (C3b-receptor; CD35), CR3 (iC3b- and beta-glucan-receptor), and CR4 (iC3b-receptor; CD11c/CD18). Studies of isolated macrophages have suggested that CR3 is the major receptor mediating capture of either C3-opsonized erythrocytes (E) or beta-glucans. In this investigation, the organ distribution and function of CR3 in the clearance of IgM-opsonized E and soluble CR3-binding polysaccharides were explored in normal vs. CR3-knockout (CR3-KO) mice. Analysis of intravenously (i.v.) injected 125I-anti-CR3 showed that the major vascular reservoir of CR3 was the liver, followed by spleen and lungs. By contrast, clearance of 125I-anti-CR1 appeared to be mediated predominantly by splenic B lymphocytes, as only subsets of splenic macrophages or Kupffer cells were found to express CR1. Clearance of IgM-opsonized 51Cr-E occurred rapidly to the livers of normal mice but was nearly absent in CR3-KO mice. Soluble 125I-beta-glucan exhibited rapid clearance to the liver in normal mice, whereas clearance in CR3-KO mice was significantly reduced. In conclusion, Kupffer cell CR3 plays a crucial role in the clearance of both IgM-opsonized E and beta-glucans.

Animals↗
PubMed1999

Beta-glucan, a "specific" biologic response modifier that uses antibodies to target tumors for cytotoxic recognition by leukocyte complement receptor type 3 (CD11b/CD18).

beta-Glucans were identified 36 years ago as a biologic response modifier that stimulated tumor rejection. In vitro studies have shown that beta-glucans bind to a lectin domain within complement receptor type 3 (CR3; known also as Mac-1, CD11b/CD18, or alphaMbeta2-integrin, that functions as an adhesion molecule and a receptor for factor I-cleaved C3b, i.e., iC3b) resulting in the priming of this iC3b receptor for cytotoxicity of iC3b-opsonized target cells. This investigation explored mechanisms of tumor therapy with soluble beta-glucan in mice. Normal mouse sera were shown to contain low levels of Abs reactive with syngeneic or allogeneic tumor lines that activated complement, depositing C3 onto tumors. Implanted tumors became coated with IgM, IgG, and C3, and the absent C3 deposition on tumors in SCID mice was reconstituted with IgM or IgG isolated from normal sera. Therapy of mice with glucan- or mannan-rich soluble polysaccharides exhibiting high affinity for CR3 caused a 57-90% reduction in tumor weight. In young mice with lower levels of tumor-reactive Abs, the effectiveness of beta-glucan was enhanced by administration of a tumor-specific mAb, and in SCID mice, an absent response to beta-glucan was reconstituted with normal IgM or IgG. The requirement for C3 on tumors and CR3 on leukocytes was highlighted by therapy failures in C3- or CR3-deficient mice. Thus, the tumoricidal function of CR3-binding polysaccharides such as beta-glucan in vivo is defined by natural and elicited Abs that direct iC3b deposition onto neoplastic cells, making them targets for circulating leukocytes bearing polysaccharide-primed CR3. Therapy fails when tumors lack iC3b, but can be restored by tumor-specific Abs that deposit iC3b onto the tumors.

Animals↗
PubMed1999

The beta-glucan-binding lectin site of mouse CR3 (CD11b/CD18) and its function in generating a primed state of the receptor that mediates cytotoxic activation in response to iC3b-opsonized target cells.

Mouse leukocyte CR3 (Mac-1, alphaMbeta2 integrin) was shown to function as a receptor for beta-glucans in the same way as human CR3. Soluble zymosan polysaccharide (SZP) or pure beta-glucans labeled with FITC or 125I bound in a saturable and reversible manner to neutrophils, macrophages, and NK cells. This lectin activity was blocked by anti-CD11b mAb M1/70 or 5C6 and did not occur with leukocytes from CR3-/- (CD11b-deficient) mice. SZP preparations containing primarily mannose or glucose bound to CR3, and the binding of 125I-labeled beta-glucan to CR3 was competitively inhibited by beta-glucans from barley or seaweed, but not by yeast alpha-mannan. Also, as with human CR3, the lectin site of mouse CR3 was inhibited by alpha- or beta-methylglucoside (but not D-glucose), alpha- or beta-methylmannoside, and N-acetyl-D-glucosamine. Phagocytosis of zymosan and serum-opsonized zymosan was partially inhibited by anti-CR3 and was reduced to <40% of normal with leukocytes from CR3-/- mice. As with neutrophils from patients with CD18 deficiency, neutrophils from CR3-/- mice exhibited no phagocytosis of particulate beta-glucan. SZP or beta-glucans primed CR3 of neutrophils, macrophages, and NK cells for cytotoxicity of iC3b-opsonized tumor cells that otherwise did not trigger killing. beta-Glucan priming for cytotoxicity was inhibited by anti-CR3 and did not occur with leukocytes from CR3-/- mice. The primed state of macrophage and NK cell CR3 remained detectable for 18 to 24 h after pulsing with beta-glucans. The similarity of mouse and human CR3 in response to beta-glucans highlights the utility of mouse tumor models for development of therapeutic beta-glucans.

Animals↗
PubMed1999

Therapeutic intervention with complement and beta-glucan in cancer.

Complement (C) has two major effector systems available for host defense. The membrane attack complex (MAC) generated from components C5-C9 can form membrane-penetrating lesions that lead to cell death by causing a rapid loss of cytoplasmic components. The MAC is only effective against pathogens with outer phospholipid membranes, and cannot kill gram-positive bacteria or yeast whose membranes are protected by cell walls. The most important effector mechanism of C is the opsonization of microbial pathogens with the serum protein C3 that leads to their high avidity attachment to the C3-receptors of phagocytic cells. Pathogens that activate complement are first coated with the C3b fragment of C3, which is rapidly proteolyzed into the iC3b fragment by serum factor I. These iC3b fragments serve to promote the high avidity attachment of the 'iC3b-opsonized' pathogens to the iC3b-receptors (CR3, CD11b/CD18) of phagocytic cells and natural killer (NK) cells, stimulating phagocytosis and/or cytotoxic degranulation. Host cells, including neoplastic tumor cells, have been endowed with natural mechanisms for self-protection against both the MAC and the cytotoxic activation of CR3. This review discusses a novel type of immunotherapy for cancer that uses soluble yeast beta-glucan to override the normal resistance of iC3b-opsonized tumor cells to the cytotoxic activation of phagocyte and NK cell CR3, allowing this important effector mechanism of the C system to function against tumor cells in the same way that it normally functions against bacteria and yeast. Moreover, the cytotoxic activation of beta-glucan-primed NK cell CR3 by iC3b-opsonized tumors is shown to be accompanied by a tumor-localized secretion of the cytokines TNFalpha, IFNalpha, IFNgamma, and IL-6.

Animals↗
PubMed1999

Anti-human procathepsin D activation peptide antibodies inhibit breast cancer development.

Enzymatically inactive procathepsin D secreted from cancer cells has been confirmed to play a role in development of human breast cancer. In the present study, we focused on the role of activation peptide which was in our preliminary studies suggested to be most probably responsible for mitogenic activity of procathepsin D. Using synthetic fragments and antibodies raised against individual fragments, we demonstrated that the growth factor activity of activation peptide is localized in a nine amino acid stretch (AA 36-44) of activation peptide and moreover both anti-activation peptide and anti-27-44 peptide antibodies administered in vivo inhibited the growth of human breast tumors in athymic nude mice. Taking into account our previous results and presented data, we hypothesize that the interaction of procathepsin D activation peptide with an unknown surface receptor is mediated by a sequence 36-44 plus close vicinity. We also propose that this interaction leads in certain types of tumor derived cell lines to proliferation and higher motility. Blocking of the interaction of activation peptide by specific antibodies or antagonists might be a valuable tool in breast cancer inhibition.

Animals↗
PubMed1998

Effect of procathepsin D and its activation peptide on prostate cancer cells.

Cathepsin D, a lysosomal aspartic proteinase, is secreted in the form of enzymatically inactive precursor in some cancer cells. This precursor, called procathepsin D, was found to exhibit growth factor activity toward breast cancer cell lines and this activity was later shown to be mediated by its activation peptide. In the present investigation we have used human procathepsin D and a synthetic 44 amino acid peptide corresponding to the activation peptide of procathepsin D to test its growth factor activity for human prostate cancer-derived cell lines PC3, DU145 and LNCaP. We have tested the level of proliferation of these cell lines depending on the presence of either procathepsin or activation peptide in the medium. In parallel, we have also measured the time dependency of this growth and established the optimal dose of activation peptide. These findings represent the first experimental data showing the direct effects of procathepsin D on prostate cancer cells.

Cathepsin D↗
PubMed1997

Analysis of the interaction of procathepsin D activation peptide with breast cancer cells.

Cathepsin D, a lysosomal aspartic proteinase, is secreted in the form of enzymatically inactive proenzyme by many types of human breast cancer tissue and exerts mitogenic activity toward these tissues. Flow cytometry was used to test the binding of procathepsin D purified from the secretion of the breast cancer cell line ZR-75-1 to human breast cancer cells. No previously known surface antigens or soluble M6P-R or anti-M6P-R antibodies were found to inhibit the specific binding of procathepsin D-FITC. Similarly, none of these potential inhibitors was found to inhibit growth factor activity of procathepsin D. Our results indicate that procathepsin D growth factor activity is mediated by a new, previously unknown receptor moiety and that the binding activity can be localized in position 27-44 of the activation peptide of procathepsin D. Furthermore, in vivo experiments indicate that treatment with anti-procathepsin D antibodies can reverse the growth of human breast tumors in athymic nude mice.

Animals↗
PubMed1997

Targeting of natural killer cells to mammary carcinoma via naturally occurring tumor cell-bound iC3b and beta-glucan-primed CR3 (CD11b/CD18).

Previous reports have suggested that malignant cells frequently generate a humoral immune response that is ineffective in tumor destruction. Despite coating tumors with IgM and IgG that activate the C system via the classical pathway, normal membrane regulators of C (e.g., membrane cofactor protein and CD59) prevent cytotoxicity. Moreover, C3 deposition on tumors does not result in cytotoxic recognition by phagocytes or NK cells bearing C3 receptors capable of mediating destruction of C3-opsonized bacteria or yeast. The current investigation showed that freshly excised mammary tumors bore IgM, IgG, and C3 detectable by flow cytometry. Normal sera contained natural IgM and IgG Abs reactive with breast tumor cell lines, and IgG Ab titers were increased in patients with breast cancer. Breast tumor cell lines incubated in normal serum from AB+ individuals activated the classical, but not the alternative, pathway of C and became coated with C3. Despite exhibiting membrane-bound C3, serum-opsonized breast tumor cell lines were not killed by CR3 (CD11b/CD18)-bearing NK cells. Priming of NK cell CR3 with small soluble yeast beta-glucan polysaccharides enabled CR3-dependent killing of these same C3-bearing tumor cell lines. Tests of mammary carcinoma cells from freshly excised tumors demonstrated that they also bore sufficient amounts of opsonic C3 for cytotoxic recognition by NK cells bearing polysaccharide-primed CR3, whereas they were largely resistant to NK cells bearing unprimed CR3. This study demonstrates the potential utility of using naturally occurring opsonic C3 on tumor cells for specific immunotherapeutic targeting by NK cells and phagocytes bearing polysaccharide-primed CR3.

Adult↗
PubMed1996

Soluble beta-glucan polysaccharide binding to the lectin site of neutrophil or natural killer cell complement receptor type 3 (CD11b/CD18) generates a primed state of the receptor capable of mediating cytotoxicity of iC3b-opsonized target cells.

When phagocyte CR3 binds to iC3b on bacteria or yeast, phagocytosis and degranulation are triggered because of simultaneous recognition of iC3b via a CD11b I-domain binding site and specific microbial polysaccharides via a lectin site located COOH-terminal to the I-domain. By contrast, when phagocyte or natural killer (NK) cell CR3 adheres to iC3b on erythrocytes or tumor cells that lack CR3-binding membrane polysaccharides, neither lysis nor cytotoxicity are stimulated. This investigation showed that soluble CR3-specific polysaccharides such as beta-glucan induced a primed state of CR3 that could trigger killing of iC3b-target cells that were otherwise resistant to cytotoxicity. Anti-CR3 added before sugars prevented priming, whereas anti-CR3 added after sugars blocked primed CR3 attachment to iC3b-targets. Polysaccharide priming required tyrosine kinase(s) and a magnesium-dependent conformational change of the I-domain that exposed the CBRM1/5 activation epitope. Unlike LPS or cytokines, polysaccharides did not up-regulate neutrophil CR3 expression nor expose the mAb 24 reporter epitope representing the high affinity ICAM-1-binding state. The current data apparently explain the mechanism of tumoricidal beta-glucans used for immunotherapy. These polysaccharides function through binding to phagocyte or NK cell CR3, priming the receptor for cytotoxicity of neoplastic tissues that are frequently targeted with iC3b and sparing normal tissues that lack iC3b.

Animals↗
PubMed1996

Function of C3 in a humoral response: iC3b/C3dg bound to an immune complex generated with natural antibody and a primary antigen promotes antigen uptake and the expression of co-stimulatory molecules by all B cells, but only stimulates immunoglobulin synthesis by antigen-specific B cells.

Previous studies have shown that an optimal humoral response to a primary protein antigen requires C3 and CR2 (CD21). Sera from non-immunized donors contain natural IgM and IgG antibodies to the primary antigen keyhole limpet haemocyanin (KLH), and these have been previously shown to form immune complexes (IC) that activate the classical pathway of C, fixing iC3b/C3dg onto the KLH antigen. Such KLH IC bind to CR2 on KLH-non-specific B lymphocytes, resulting in antigen processing and MHC class II-dependent presentation to KLH-specific helper T cells. KLH IC also induce B lymphocytes to express the CD80 costimulatory molecule via simultaneous CR2 ligation with C3 and Fc gammaRII (CD32) stimulation by IgG natural antibody. The current study demonstrated that KLH IC ligation to either CR2 or Fc gammaRII resulted in activation of a second co-stimulatory molecule, LFA-1 (CD11a, CD18). The possibility of polyclonal B cell stimulation by the presentation of KLH-iC3b/C3dg by antigen-non-specific B cells was excluded by demonstration that in vitro cultivation of peripheral blood mononuclear cells (PBMC) with KLH-iC3b/C3dg elicited only anti-KLH, and did not stimulate synthesis of antibodies to hepatitis C virus (HCV) or tetanus toxoid (TT). Of greatest significance, a specific anti-KLH response was only detectable in cultures stimulated with KLH-iC3b/C3dg and not in cultures stimulated with KLH alone or KLH-IgG. Thus, iC3b/C3dg that was bound to a primary protein antigen enhanced recognition and specific immunoglobulin synthesis by antigen-specific B cells, even though the antigen was taken up and processed via CR2 by both antigen-specific and non-specific B cells.

Antibodies, Monoclonal↗
PubMed1995

Human procathepsin D: three-dimensional model and isolation.

Human procathepsin D was isolated from medium of human breast cancer cell line ZR-75-1 potentiated with estrogen. The isolation involved both immunoaffinity chromatography and ion-exchange chromatography. The affinity chromatography employed polyclonal antibodies raised against a synthetic activation peptide of human cathepsin D. We have started preliminary crystallization trials using the isolated material. A model of human procathepsin D was also built using coordinates of human cathepsin D and pig pepsinogen. The model aids understanding of multiple roles played by activation peptides of aspartic proteinases and will be used as a starting model for molecular replacement.

Amino Acid Sequence↗
PubMed1994

Mitogenic function of human procathepsin D: the role of the propeptide.

The proform of cathepsin D is secreted by some human breast-cancer cell lines upon stimulation with oestrogen. In these cell lines, procathepsin D was described to act as an autocrine mitogen, and a correlation between the cathepsin D concentration in tumour tissues and poor prognosis for the patient was demonstrated in several independent investigations. In the present study, we focused on the mechanism of procathepsin D mitogenic activity. Procathepsin D isolated from secretions of ZR-75-1 breast-cancer cell line was used to test for mitogenic activity on a set of seven human cell lines. For nanomolar procathepsin D concentrations, we found a stronger dose-responsive cellular reaction in the case of several different human breast-cancer-derived cell lines. The mitogenic activity was not blocked by the inhibition of proteolytic activity nor by the inhibition of the interaction of procathepsin D with mannose-6-phosphate receptors. On the other hand, the addition of antibodies raised against the propeptide impaired the mitogenic activity of procathepsin D, and a synthetic peptide alone corresponding to the propeptide of procathepsin D produced similar effects, as did the zymogen molecule. The synthetic propeptide was shown to block partially the interaction of procathepsin D with the cellular surface. Our results indicate that the mitogenic function involves the propeptide of cathepsin D, which appears to be recognized by a surface receptor.

Cathepsin D↗
PubMed1993

Effect of age on antibody responses in low responder C57BL/10ScSn and high responder A/J strains of mice.

The IgM antibody response to sheep red blood cells (SRBC) appeared significantly earlier in A/J strain mice (already in 10-day-old animals), but after 21 days responses were higher in B10 mice. These differences disappeared after reaching adulthood and IgM responses after either primary or secondary immunization were thereafter comparable in these strains. High-responder A/J mice made significantly more IgG antibodies than low-responder B10 mice from 21 days of age and strong differences lasted until the age of 19 months, when IgG antibody production was again similar. Potentiation of IgM formation by simultaneous application of 10 micrograms of LPS was higher in B10 mice until 19 months of age. On the other hand, potentiation of the IgG response was markedly high in B10 mice only in adult animals (3 months). Thereafter the potentiation was higher in A/J mice. The onset of Ig secretion in A/J mice was at 15 days and markedly increased at the age of 30 days. Levels of immunoglobulin synthesis remained extremely low in B10 mice. Age-related changes in IgG antibody production generally correlated with the decline of MHC class II antigen expression on peritoneal macrophages in these strains.

Aging↗