Tatjana Srdic-Rajic

TNF-α Induced Apoptosis is Accompanied with Rapid CD30 and Slower CD45 Shedding from K-562 Cells

TNF-α can induce cell death (apoptosis and necrosis), and these effects mostly depend on expression of TNF-receptor superfamily molecules. As determination of certain intracellular enzymes like LDH, released from cultured tumor cells, reflects early membrane alterations, we compared LDH release with changes in cell surface membrane molecule expression during culture of K-562 cells in the presence of TNF-α. TNF-α-mediated CD45 and CD30 shedding is shown to be to be time- and dose-dependent and associated with significant increase in LDH release, with maximal effects after 24xa0h of treatment. The percentage of decrease of all examined cell surface molecules on K-562 cells after TNF-α treatment was not uniform and appeared to depend on the respective constitutive level of expression and molecule type. The presence of these molecules was confirmed in supernatants using Western blot analyses. These results indicated the complexity of events on the cell membrane, including early LDH release that is associated with a difference in shedding of CD30 and CD45. Shedding of CD30 occurs before apoptosis induction, while shedding of CD45 is associated with apoptosis.

Decreased expression of pSTAT, IRF-1 and DAP10 signalling molecules in peripheral blood lymphocytes of patients with metastatic melanoma

Aims As numerous signalling molecules regulate effector functions of peripheral blood lymphocytes (PBLs) that have an important anti-tumour activity, the aim of this study was to analyse their level in patients with metastatic melanoma (MM) compared with healthy controls (HCs). Methods Peripheral blood mononuclear cells (PBMCs) of 36 MMs and 28 HCs were analysed for the level of perforin, interferon-regulating transcription factor-1 (IRF-1), DAP10 and Src homology 2 domain-containing tyrosine phosphatase-1 by reverse transcriptase PCR, level of phosphorylated signal transducers and activators of transcription (pSTAT)-1, pSTAT-4, pSTAT-5 by western blot and interferon (IFN)-γ production by ELISA. The expression of activating NKG2D and inhibitory killer immunoglobulin-like receptors (KIR), CD158a and CD158b, on PBL, CD3−CD56+ natural killer (NK) cells and CD3+CD8+ cytotoxic T lymphocytes (CTLs), as well as the percentage of CD14+HLA-DR- cells in PBMC were estimated by flow cytometry. Results Patients with MM, compared with HCs, had significantly lower level of cytotoxic molecule perforin and decreased IFN-γ production, as well as lower level of pSTAT-1, pSTAT-4, pSTAT-5 and IRF-1 signalling molecules in PBMC. Furthermore, MM had decreased expression of activating NKG2D receptor on PBL and NK cells and low level of its DAP10 signalling molecule contrary to no changes in KIR expression on all investigated cells. These results could be associated with increased percentage of immunosuppressive CD14+HLA-DR− myeloid-derived suppressor cells detected in patients with MM. Conclusions The altered signalling molecules of PBL could represent biomarkers of impaired cytotoxic and immunoregulatory function of these cells, indicating melanoma-associated immunosuppression that facilitates tumour progression.

Naturally occurring V region connected antibodies inhibit anti-dsDNA antibody reactivity with dsDNA.

The production of autoantibodies against a vast array of self antigens, most notably double stranded (ds) DNA, characterized systemic lupus erythematosus (SLE). The purpose of this work is to study specific Ig fractions isolated from normal human serum (NHS) and their effect on the binding of anti-double-stranded deoxyribonucleic acid (dsDNA) antibodies (Abs) to dsDNA. A fraction named immunoglobulin G (IgG)-reactive IgG was purified from total NHS IgG by absorption onto (CNBr)-activated Sepharose 4B linked to intact IgG molecules (IgG-Sepharose column). IgG-reactive IgG was co-incubated with systemic lupus erythematosus (SLE) patients serum and binding of the anti-dsDNA Abs to dsDNA was measured by enzyme-linked immunosorbent assay (ELISA). Co-incubation of SLE patients serum with IgG-reactive IgG resulted in a dose-dependent reduction in binding of anti-dsDNA Abs to dsDNA. A reduction greater than 70% was observed at a concentration of 300 μg of IgG-reactive IgG per mL of a 400-fold diluted SLE patients serum whereas total NHS IgG, at the same concentration, resulted in a 10% reduction in binding. The purification process used to isolate IgG-reactive IgG was based on interactions between intact Ig rather than on interactions between F(ab)(2) portions. IgG(2) is the predominant immunoglobulin (Ig) subclass in IgG-reactive IgG. Thus, IgG(2) might have an important role in the connectivity characteristics of NHS IgG. The capacity of IgG-reactive IgG to inhibit anti-DNA Ab binding to dsDNA may have potential application in the treatment of SLE. This targeted biological approach may provide an alternative strategy to immunosuppressants.

Synthesis and in vitro antitumour activity of crassalactone D, its stereoisomers and novel cinnamic ester derivatives

Naturally occurring styryl lactone, crassalactone D (1), unnatural 4-epi-crassalactone D (2), and the corresponding 7-epimers (3 and 4) have been synthesized starting from d-glucose. The key step of the synthesis is a new one-pot sequence that commenced with a Z-selective Wittig olefination of suitably functionalized sugar lactols with a stabilized ylide, (methoxycarbonylmethylene)-triphenylphosphorane, in dry methanol, to afford 1 or 3, in the mixtures with the corresponding 4-epimers (2 or 4, respectively). A number of 6-O-cinnamoyl derivatives of styryl lactones 1-4 have been prepared, bearing electron donating or electron withdrawing functionalities in the C-4 position of cinnamic acid residue. The synthesized products were evaluated for their inxa0vitro antiproliferative activity against selected human tumour cell lines, whereupon very potent cytotoxicities have been recorded in many cases. SAR analysis indicated some important structural features responsible for biological activity, such as stereochemistry at the C-4 and C-7 positions, as well as the nature of a substituent at the C-4 position in the aromatic ring of cinnamoate moiety. Flow cytometry and Western blot analysis data gave insight in the mechanism underlying antiproliferative effects of the synthesized compounds.

Novel O-methyl goniofufurone and 7-epi-goniofufurone derivatives: Synthesis, in vitro cytotoxicity and SAR analysis

Novel goniofufurone (1) and 7-epi-goniofufurone (2) derivatives bearing a methoxy group at the C-5 and/or C-7 positions were prepared and their in vitro antitumour activity against some human tumour cell lines was evaluated. Some of the analogues displayed powerful antiproliferative effects against the studied tumour cells, but almost all of them were non-cytotoxic toward the normal cells (MRC-5). A SAR study reveals that the introduction of a methoxy group at the C-7 position may increase the antiproliferative effects of the analogues. The most active compounds are 7-O-methyl derivatives of goniofufurone (3) and 7-epi-(+)-goniofufurone (6), which exhibited 1177- and 451-fold higher potencies than the leads 1 and 2 toward the MDA-MB 231 cell line. At the same time, compound 3 is almost 1.5-fold more active than the commercial drug doxorubicin (DOX) against the same cell line. Flow cytometry data confirmed that the cytotoxic effects of these analogues are mediated by apoptosis, additionally revealing that these molecules induced changes in the K562 cell cycle distribution.

Antibody Epitope Specificity for dsDNA Phosphate Backbone Is an Intrinsic Property of the Heavy Chain Variable Germline Gene Segment Used

Analysis of protein sequences by the informational spectrum method (ISM) enables characterization of their specificity according to encoded information represented with defined frequency (F). Our previous data showed that F(0.367) is characteristic for variable heavy chain (VH) domains (a combination of variable (V), diversity (D) and joining (J) gene segments) of the anti-phosphocholine (PC) T15 antibodies and mostly dependent on the CDR2 region, a site for PC phosphate group binding. Because the T15 dsDNA-reactive U4 mutant also encodes F(0.367), we hypothesized that the same frequency may also be characteristic for anti-DNA antibodies. Data obtained from an analysis of 60 spontaneously produced anti-DNA antibody VH domain sequences supported our hypothesis only for antibodies, which use V gene segment in germline configuration, such as S57(VH31), MRL-DNA22, and VH11, members of the VH1 (J558) and VH7 (S107) gene families. The important finding is that out of seven V gene segments used by spontaneous anti-DNA antibodies, F(0.367) is only expressed by the germline configuration of these three V gene segments. The data suggest that antibody specificity for the phosphate group moiety delineated as F(0.367) is the intrinsic property of the V germline gene segments used, whereas paratope/epitope interaction with antigens bearing this epitope, such as PC or dsDNA, requires corresponding antibody VH conformation that is susceptible to somatic mutation(s).

Benzothiazole carbamates and amides as antiproliferative species

A series of new benzothiazole-based carbamates and amides were synthesized and their antiproliferative activity was determined. Derivatives with profound activity were identified and further investigated for their possible mechanism of action. It was found that these compounds induce specific apoptosis, G2/M cell cycle arrest and decrease ROS level in MCF-7 human breast cancer cell line. Moreover, submicromolar antiproliferative activity of examined carbamates against NT2/D1 testicular embryonal carcinoma was shown. The most potent derivatives strongly inhibited NT2/D1 cell migration and invasiveness.