Gordana Konjevic

Activity of MMP-2 and MMP-9 in sera of breast cancer patients

Gelatinase A (MMP-2) and gelatinase B (MMP-9) are proteolytic enzymes involved in the process of tumor invasion, and they are considered as possible tumor markers in breast cancer patients. In this study, we examined serum activity of proMMP-2 and proMMP-9 in relation to TNM stage, tumor size, lymph node involvement, grade of differentiation of tumors, as well as steroid and Her2/neu receptor status in breast cancer patients. The activity of gelatinase in the sera of 52 patients was analyzed by SDS-PAGE zymography. The activity of proMMP-2 and proMMP-9 significantly increased with each advancing clinical stage of disease (p=0.02-0.0009) and compared to controls (p=0.015 to p<0.01). We found a positive correlation between the activity of proMMP-2 and proMMP-9 and tumor size (p=0.007; p=0.05). Patients with lymph node-positive cancer have higher proMMP-2 and proMMP-9 activity than those with node-negative cancer. ProMMP-2 and proMMP-9 activity is not associated with the expression of Her2/neu receptors, but patients with Her2/neu overexpression (3+) showed increased proMMP-2 activity. Steroid receptor score is not associated with enhanced gelatinase activity. The relationship between the increase in proMMP-2 and proMMP-9 activity in serum and tumor size and lymph node status suggests the usefulness of these enzymes as staging markers of breast cancer patients.

Investigation of NK cell function and their modulation in different malignancies

NK cells have become a subject of investigation not only in the field of tumor immunology and infectious diseases, but also within all aspects of immunology, such as transplantation, autoimmunity, and hypersensitivity. Our early studies aside from investigating NK cell activity in experimental animals and humans included studies of perforin expression and modulation in this lymphocyte subset. As NK cell activity is modified by their environment, we showed clinical stage–dependent impairment of their activity and in vitro effect of different sera, Th1 cytokines, and their combination in breast cancer, Hodgkin’s disease, and non-Hodgkin’s lymphoma patients, especially with respect to metabolic and cell membrane changes of peripheral blood lymphocytes evaluated by spontaneous release of the enzyme lactate dehydrogenase (LDH) that led to the correction of the LDH enzyme release assay for natural cytotoxicity. By long-term immuno-monitoring of patients with malignancies, we also showed the kinetics of NK cell modulation during chemo-immunotherapy. In our more recent studies, we give data of NK function and novel families of NK cell receptor expression in healthy individuals that may be of help in NK cell profiling, by giving referent values of basic and cytokine-induced expression of some NK cell receptors either in evaluation of disease or in immuno-monitoring during cytokine therapy of patients with malignancies. Moreover, we give novel aspects of modulation of NK cell activity by cytokines approved for immunotherapy, IFN and IL-2, in melanoma and other malignancies with respect to alterations in new activating (NKG2D and CD161) and inhibitory (CD158a and CD158b) receptor characteristics and signaling molecules in CD16- and CD56-defined NK cells and their small immunoregulatory and large cytotoxic subsets in peripheral blood and lymph nodes, as NK cell-mediated killing of tumor cells depends on the balance between stimulatory and inhibitory signaling.

Values of MMP-2 and MMP-9 in Tumor Tissue of Basal-Like Breast Cancer Patients

Gelatinase A (MMP-2) and gelatinase B (MMP-9) are proteolytic enzymes involved in process of tumor invasion, and they are considered as possible tumor markers in breast cancer patients. In this study, we measured activity of latent and active form of MMP-2 and MMP-9 in tumor and adjacent tissue of 60 breast cancer patients by SDS-PAGE zymography. The activity of both form of gelatinases significantly increased with each advancing clinical stage of disease. ProMMP-9 and aMMP-9 activity in tumor tissue shows a positive association with tumor size. Patients with lymph node involvement have higher proMMP-2, aMMP-2 and aMMP-9 activity than node negative patients. Steroid receptor-negative tumors had enhanced aMMP-2 and aMMP-9 activity. Patients with basal-like cancers had higher proMMP-2 tumor activity and aMMP-2 adjacent tissue activity compared to patients with luminal A tumors. Patients with negative hormone receptors are associated with increased activity of both form of gelatinases in adjacent tissue. Reported increased activity of MMP-2 in tumor and adjacent tissue of basal-like tumors implicates that MMP-2 might have a role in aggressive biology of basal-like cancers. Additional investigations regarding molecular pathways in adjacent tissue could give better insight into aggressive nature of basal-like carcinomas.

Lactate dehydrogenase, Catalase, and Superoxide dismutase in Tumor Tissue of Breast Cancer Patients in Respect to Mammographic Findings

Lactate dehydrogenase (LDH), marker of anaerobic metabolism, is associated with highly invasive and metastatic breast cancer. Novel studies show that increased anaerobic metabolism (LDH), as well as activity of antioxidative enzymes (superoxide dismutase (SOD) and catalase (CAT)), is correlated with higher mammographic density, as known predictor of breast cancer risk. In this study, we measured LDH, MDH, and SOD activity in tumor and adjacent tissues of breast cancer patients by spectrophotometric assay. Mammograms were evaluated according to the American College of Radiology Breast Imaging Reporting and Data system. Mammographically dense breast tissue is associated with higher activity of LDH in tumor tissue of breast cancer patients. Moreover, patients with masses have significantly higher activity of LDH compared to patients with focal asymmetries or architectural distortion. Patients with spiculated mass margin had higher activity of LDH compared to patients with focal asymmetries or architectural distortion. Activity of LDH in patients significantly increases, while activity of CAT significantly decreases with the increase of BIRADS category. These results suggest that the association of activity of LDH and CAT in tumor tissue with mammographic characteristics could help in defining aggressive breast cancers.

Distribution of several activating and inhibitory receptors on CD3-CD16+ NK cells and their correlation with NK cell function in healthy individuals.

The aim of this study was to estimate the distribution and density of a representative set of activating and inhibitory receptors on gated natural killer (NK) cells, as well as on their bright and dim subsets, and to correlate the receptor expression with NK cell activity for healthy individuals on CD3−CD16+ NK cells. We show that in 43 healthy controls NK cell activity against K562 target cells was 37.34% (E:T, 80:1) by standard chromium release assay. The expression of receptors on NK cells and their subsets was analyzed by flow cytometry. The cytotoxic CD3−CD16bright NK subset constituted 78.97%, while the regulatory CD3−CD16dim NK subset constituted 21.03% of NK cells. We show the distribution of NKG2D, CD161, CD158a, and CD158b receptors on CD3−CD16+ NK cells in peripheral blood lymphocytes (PBLs), on gated NK cells, and on the CD3−CD16bright and CD3−CD16dim subsets. Contrary to CD158a and CD158b killer immunoglobulin-like receptors (KIRs), there is a significant positive correlation of NKG2D and CD161 expression with NK cytotoxicity. We show the kinetics of change in CD3−CD16+NK/K562 conjugate composition, together with the stronger target binding capacity of CD16bright NK cells. Furthermore, we show that after coculture of PBLs with K562 the expression of CD107a, a degranulation marker, on CD3−CD16+NK cells and subsets is time dependent and significantly higher on the cytotoxic CD3−CD16bright NK subset. The novel data obtained regarding expression of NK cell activating and inhibitory receptors for healthy individuals may aid in detecting changes that are associated with various diseases.

The stage dependent changes in NK cell activity and the expression of activating and inhibitory NK cell receptors in melanoma patients.

BACKGROUND As NK cell antitumor activity is regulated by the balance between numerous activating and inhibitory receptors the aim of this study was to analyze the changes in these receptors expression as well as the differences in their association with NK cell activity in melanoma patients in different clinical stages. MATERIALS AND METHODS Forty-three melanoma patients in the three different clinical stages and 17 healthy controls were analyzed for NK cell activity by the standard (51)Cr radioactive assay, as well as for the percentage and absolute number of CD3-CD56+ NK cells and their subsets and the expression of degranulation marker CD107a, activating NKG2D and CD161, and inhibitory KIR, CD158a, and CD158b receptors on CD3-CD56+ NK cells by flow cytometry. IFN-γ and TNF-α PBL production were evaluated by enzyme-linked immunosorbent assay (ELISA). RESULTS We show that contrary to nonmetastatic, metastatic melanoma patients have significantly impaired NK cell activity, lower CD107a expression, as well as decreased production of IFN-γ and TNF-α. Furthermore, these melanoma patients have a significant decrease in the expression of activating NKG2D receptor, which positively correlates with NK cytotoxicity, and a significant increase in the expression of inhibitory CD158b on CD3-CD56+ NK cells compared with healthy controls. CONCLUSIONS In this study, we show that metastatic melanoma patients have significant changes in NK cell activity and the expression of activating as well as inhibitory NK cell receptors. These results indicate that only in this patient group, the blocking of inhibitory or enhancing of activating NK cell receptor expression may promote NK cell-mediated cytolysis.

Novel aspects of in vitro IL-2 or IFN-α enhanced NK cytotoxicity of healthy individuals based on NKG2D and CD161 NK cell receptor induction

As IL-2 and IFN-α modulate NK cell activity it was of interest to investigate the expression of newly defined NK cell receptors and augmented NK cell activity in healthy individuals after cytokine in vitro treatment. Peripheral blood lymphocytes (PBL) obtained from 31 healthy volunteers treated for 18 h with 200 IU/ml IL-2 and 250 IU/ml IFN-α were evaluated for NK cell cytotoxicity. Expression of NKG2D, CD161, CD158a, CD158b receptors was analyzed on CD3⁻CD16+ NK cells, cytotoxic CD16(bright) and regulatory CD16(dim) subsets by FACS flow. The found induced significant in vitro enhancement of NK cell activity by both cytokines is supported by specific cytokine induction in PBL of pSTAT1 and pSTAT5, determined by Western blotting, as well as induction of IRF-1 transcription. Both cytokines induce significant up-regulation of NKG2D expression while only IFN-α induced significant up-regulation of CD161, with no alteration in KIR expression by either cytokine on CD3⁻CD16+ NK cells. Investigated cytokines did not induce change in NK cell bright and dim subset distribution. Moreover, we find that, not only cytokine receptor induction on the CD3⁻CD16+ NK cells, but also simultaneous increase in their percentage and/or density on CD16(bright) and CD16(dim) subsets, represent good indicators of receptor cytokine-susceptibility. As the role of NK cells has been shown in the loss of tolerance, infection and cancer, the data obtained in this study may be of help in NK cell profiling, by giving referent values of cytokine-induced novel NK cell receptor expression either in evaluation of these diseases or in immunomonitoring during cytokine immunotherapy.

Distribution of several activating and inhibitory receptors on CD3−CD56+ NK cells in regional lymph nodes of melanoma patients

BACKGROUND Natural killer (NK) cells, as the main effector subpopulation of the innate immune system, play an important role in the control of the rise and spread of malignant tumors. Regional lymph nodes (LN) represent the first immunologic barrier to tumor metastasis. Since there are scarce data on NK cells from regional LN of cancer patients, the aim of this study was to investigate the expression of several activating and inhibitory receptors on the entire NK cell population as well as their CD3(-)CD56(dim) and CD3(-)CD56(bright) functional NK subsets from regional LN of melanoma patients. MATERIALS AND METHODS Mononuclear cells were isolated from 50 regional LN of melanoma patients. The expression of several receptors on NK cells and their functional subsets was analyzed by flow cytometry. RESULTS We show increased percentages of CD3(-)CD56(+) NK cells in involved LN compared with uninvolved LN, mostly in favor of the CD56(dim) NK cell subset. NK cells in involved LN express similar levels of activating receptor NKG2D, while the level of another activating receptor, CD16, is increased compared with uninvolved LN. Regarding the expression of inhibitory NK cell receptors, we show increased CD158b, but similar low CD158a, inhibitory killer Ig-like cell receptor expression in involved LN compared with uninvolved LN. Furthermore, NK cells in involved compared with uninvolved LN displayed increased CD69 early activation antigen expression. CONCLUSIONS Our results indicate that with tumor infiltration into regional LN of melanoma patients, NK cells, mostly of the CD56(dim) subset, are recruited into draining LN. The invading NK cells show counterbalance of the increased expression of CD16 activating receptor and increased CD158b inhibitory killer Ig-like cell receptor.

CD69 on CD56+ NK cells and response to chemoimmunotherapy in metastatic melanoma

Background  The few chemoimmunotherapy trials that together with dacarbazine (DTIC) and interferon‐α 2a (IFNα), include retinoic acid (RA), did not include detailed immunological evaluation of functional and phenotypic natural killer (NK) cell characteristics, and have shown contradictory clinical results.

Antiproliferative Effect of 13-cis-Retinoic Acid is Associated with Granulocyte Differentiation and Decrease in Cyclin B1 and Bcl-2 Protein Levels in G0/G1 Arrested HL-60 Cells

Retinoic acid (RA), similar to specific growth factors, can induce differentiation of proliferating promyelocytic precursors into terminally differentiated granulocytes, although little is known about effects of its 13-cis isomer on promyelocytic leukemia (PML). In this study we demonstrate that 13-cis-RA has a dose and time-dependant antiproliferative effect on HL-60 PML cell line, that it induces cell accumulation in resting G0/G1 phase of the cell cycle followed by an increase in CD11b granulocyte differentiation antigen expression. The obtained increase in the percentage of HL-60 cells in G0/G1 phase and complementary decrease in S phase of the cell cycle are accompanied by a decrease in the expression of cell cycle regulatory molecule cyclin B1. We also show the induction of interferon regulatory factor-1 (IRF-1) transcription that can, also, to some extent contribute to the antiproliferative effect of 13-cis-RA. Furthermore, down-regulation of Bcl-2 protein expression in 13-cis-RA treated HL-60 cells may contribute to sensitivity to apoptosis of growth arrested HL-60 promyelocytic cells.