The profile of lysosomal exoglycosidases in replicative and stress-induced senescence in early passage human fibroblasts

The aim of the present study was to assess the profiles of the exoglycosidases: N-acetyl-β-hexosoaminidase, β glucuronidase and β galactosidase, α mannosidase and α fucosidase in fibroblast culture undergoing replicative and stress-induced senescence. Half of the cell culture was grown in normal conditions, without the stressor, and the other half of the cell was treated with 0.15 mM tert-butylhydroperoxide. The activities of total N-acetyl-β-hexosoaminidase as well as β glucuronidase in the cell lysate were determined in duplicates using the method of Marciniak et al. The activities of β galactosidase, α mannosidase and α fucosidase in the cell lysate were determined in duplicates using the method of Chatteriee et al. with the modification by Zwierz et al. The activities of the exoglycosidases examined, with the exception of β glucuronidase, showed a significant increase between individual days of the experiment in both non-stressed and stressed fibroblast cell culture. On each day of the experiment, in the cell lysate of stressed fibroblasts, the activities of exoglycosidases were significantly higher compared to the non-stressed cells. There were very strong correlations between SA-β-GAL staining and b galactosidase activity on individual days of the experiment in both non-stressed and stressed fibroblast cell culture. Replicative and stress-induced senescence results in significant changes to the level of lysosomal exoglycosidases, and results in enhanced lysosomal degradative capacity.     

Wnt3a binds to several sFRPs in the nanomolar range

Secreted Frizzled-related proteins (sFRPs) are modulators of the Wnt signaling pathway that plays important roles in both embryogenesis and oncogenesis. sFRPs have been proposed to antagonize Wnt activity by binding to Wnts. However, the affinity of this binding is unknown. Here we show, using surface plasmon resonance and purified proteins, that sFRP1, sFRP2, sFRP4, and Frzb bind directly to Wnt3a with affinities in the nanomolar range. However, only sFRP1 and sFRP2 antagonize Wnt3a activity by blocking Wnt3a induced β-catenin accumulation in L cells. Furthermore, sFRP2, but not Frzb, antagonizes Wnt3a signaling in an ES cell model of mesoderm differentiation. These results provide the first measurement of binding affinity of sFRPs for a Wnt, which together with the measurement of antagonistic activity of sFRPs could help understand how sFRPs regulate Wnt signaling.     

CD40L and IL-4 stimulation of acute lymphoblastic leukemia cells results in upregulation of mRNA level of FLICE--an important component of apoptosis

The use of cancer vaccines based on dendritic cells (DC) presenting tumor antigens can be a promising tool in the treatment of leukemia. The functional characteristics of leukemia derived DC is still to be elucidated. CD40 promotes survival, proliferation and differentiation of normal B cells. CD40 triggering was used to enhance the poor antigen-presenting capacity of leukemic B-cells. Since it is still unclear whether CD40 ligation drives neoplastic B-cells to apoptosis or not, we assessed the mRNA expression of FLICE, FAS, FADD and TRADD - important components of apoptosis machinery, using real-time PCR in acute lymphoblastic leukemia cells before and after CD40 and IL-4 stimulation. ALL cells stimulated with CD40L/IL-4 expressed dendritic cell phenotype at mRNA and protein levels (upregulation of main costimulatory and adhesion molecules noted in real-time RT PCR and flow cytometry); they also expressed higher amounts of mRNA for FLICE, TRADD and FADD after CD40L/IL-4 stimulation. However differences statistically significant comparing cells cultured with CD40L/IL-4 and medium alone regarded only FLICE. Concluding, we showed upregulation of important elements of apoptosis at mRNA level in ALL cells after CD40 ligation.     

Induction of protective immune responses against NXS2 neuroblastoma challenge in mice by immunotherapy with GD2 mimotope vaccine and IL-15 and IL-21 gene delivery

The GD2 ganglioside expressed on neuroectodermal tumor cells is weakly immunogenic in tumor-bearing patients and induces predominantly IgM antibody responses in the immunized host. Using a syngeneic mouse challenge model with GD2-expressing NXS2 neuroblastoma, we investigated novel strategies for augmenting the effector function of GD2-specific antibody responses induced by a mimotope vaccine. We demonstrated that immunization of A/J mice with DNA vaccine expressing the 47-LDA mimotope of GD2 in combination with IL-15 and IL-21 genes enhanced the induction of GD2 cross-reactive IgG2 antibody responses that exhibited cytolytic activity against NXS2 cells. The combined immunization regimen delivered 1 day after tumor challenge inhibited subcutaneous (s.c.) growth of NXS2 neuroblastoma in A/J mice. The vaccine efficacy was reduced after depletion of NK cells as well as CD4⁺ and CD8⁺ T lymphocytes suggesting involvement of innate and adaptive immune responses in mediating the antitumor activity in vivo. CD8⁺ T cells isolated from the immunized and cured mice were cytotoxic against syngeneic neuroblastoma cells but not against allogeneic EL4 lymphoma, and exhibited antitumor activity after adoptive transfer in NXS2-challenged mice. We also demonstrated that coimmunization of NXS2-challenged mice with the IL-15 and IL-21 gene combination resulted in enhanced CD8⁺ T cell function that was partially independent of CD4⁺ T cell help in inhibiting tumor growth. This study is the first demonstration that the mimotope vaccine of a weakly immunogenic carbohydrate antigen in combination with plasmid-derived IL-15 and IL-21 cytokines induces both innate and adaptive arms of the immune system leading to the generation of effective protection against neuroblastoma challenge. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by the Roswell Park Alliance Foundation, funds to commemorate Dr. Goro Chihara’s research activity, and by a research grant R21 AI060375 from the National Institutes of Health.     

Molecular functions of the iron-regulated metastasis suppressor,NDRG1, and its potential as a molecular target for cancer therapy

N-myc down-regulated gene 1 (NDRG1) is a known metastasis suppressor in multiple cancers, being also involved in embryogenesis and development, cell growth and differentiation, lipid biosynthesis and myelination, stress responses and immunity. In addition to its primary role as a metastasis suppressor, NDRG1 can also influence other stages of carcinogenesis, namely angiogenesis and primary tumour growth. NDRG1 is regulated by multiple effectors in normal and neoplastic cells, including N-myc, histone acetylation, hypoxia, cellular iron levels and intracellular calcium. Further, studies have found that NDRG1 is up-regulated in neoplastic cells after treatment with novel iron chelators, which are a promising therapy for effective cancer management. Although the pathways by which NDRG1 exerts its functions in cancers have been documented, the relationship between the molecular structure of this protein and its functions remains unclear. In fact, recent studies suggest that, in certain cancers, NDRG1 is post-translationally modified, possibly by the activity of endogenous trypsins, leading to a subsequent alteration in its metastasis suppressor activity. This review describes the role of this important metastasis suppressor and discusses interesting unresolved issues regarding this protein.     

Steroidal saponins from the genus <Emphasis Type="Italic">Allium</Emphasis>

Steroidal saponins are widely distributed among monocots, including the Amaryllidaceae family to which the Allium genus is currently classified. Apart from sulfur compounds, these are important biologically active molecules that are considered to be responsible for the observed activity of Allium species, including antifungal, cytotoxic, enzyme-inhibitory, and other. In this paper, literature data concerning chemistry and biological activity of steroidal saponins from the Allium genus has been reviewed.     

Peptidyl Arginine Deiminase from Porphyromonas gingivalis Abolishes Anaphylatoxin C5a Activity

Evasion of killing by the complement system, a crucial part of innate immunity, is a key evolutionary strategy of many human pathogens. A major etiological agent of chronic periodontitis, the Gram-negative bacterium Porphyromonas gingivalis, produces a vast arsenal of virulence factors that compromise human defense mechanisms. One of these is peptidylarginine deiminase (PPAD), an enzyme unique to P. gingivalis among bacteria, which converts Arg residues in polypeptide chains into citrulline. Here, we report that PPAD citrullination of a critical C-terminal arginine of the anaphylatoxin C5a disabled the protein function. Treatment of C5a with PPAD in vitro resulted in decreased chemotaxis of human neutrophils and diminished calcium signaling in monocytic cell line U937 transfected with the C5a receptor (C5aR) and loaded with a fluorescent intracellular calcium probe: Fura-2 AM. Moreover, a low degree of citrullination of internal arginine residues by PPAD was also detected using mass spectrometry. Further, after treatment of C5 with outer membrane vesicles naturally shed by P. gingivalis, we observed generation of C5a totally citrullinated at the C-terminal Arg-74 residue (Arg74Cit). In stark contrast, only native C5a was detected after treatment with PPAD-null outer membrane vesicles. Our study suggests reduced antibacterial and proinflammatory capacity of citrullinated C5a, achieved via lower level of chemotactic potential of the modified molecule, and weaker cell activation. In the context of previous studies, which showed crosstalk between C5aR and Toll-like receptors, as well as enhanced arthritis development in mice infected with PPAD-expressing P. gingivalis, our findings support a crucial role of PPAD in the virulence of P. gingivalis.