Claudia Gemetzi

Potent inhibitory action of red wine polyphenols on human breast cancer cells.

Breast cancer (one of the most common malignancy in Western societies), as well as esophagus, stomach, lung, bladder, and prostate cancer, depend on environmental factors and diet for growth and evolution. Dietary micronutriments have been proposed as effective inhibitory agents for cancer initiation, progression, and incidence. Among them, polyphenols, present in different foods and beverages, have retained attention in recent years. Red wine is a rich source of polyphenols, and their antioxidant and tumor arresting effects have been demonstrated in different in vitro and in vivo systems. In the present study, we have measured the antiproliferative effect of red wine concentrate, its total polyphenolic pool, and purified catechin, epicatechin, quercetin, and resveratrol, which account for more than 70% of the total polyphenols in red wine, on the proliferation of hormone sensitive (MCF7, T47D) and resistant (MDA‐MB‐231) breast cancer cell lines. Our results indicate that polyphenols, at the picomolar or the nanomolar range, decrease cell proliferation in a dose‐ and a time‐dependant manner. In hormone sensitive cell lines, a specific interaction of each polyphenol with steroid receptors was observed, with IC50s lower than previously described. Interaction of polyphenols with steroid receptors cannot fully explain their inhibitory effect on cell proliferation. In addition, discrete antioxidant action on each cell line was detected under the same concentrations, both by modifying the toxic effect of H2O2, and the production of reactive oxygen species (ROS), after phorbol ester stimulation. Our results suggest that low concentrations of polyphenols, and consecutively, consumption of wine, or other polyphenol‐rich foods and beverages, could have a beneficial antiproliferative effect on breast cancer cell growth. J. Cell. Biochem. 78:429–441, 2000.

Wine antioxidant polyphenols inhibit the proliferation of human prostate cancer cell lines.

The effect of different wine antioxidant polyphenols (catechin, epicatechin, quercetin, and resveratrol) on the growth of three prostate cancer cell lines (LNCaP, PC3, and DU145) was investigated. A dose- and time-dependent inhibition of cell growth by polyphenols was found at nanomolar concentrations. The proliferation of LNCaP and PC3 cells was preferentially inhibited by flavonoids (catechin, epicatechin, and quercetin), whereas resveratrol was the most potent inhibitor of DU145 cell growth. Possible mechanisms of action were investigated: 1) The competition of polyphenols for androgen binding in LNCaP cells revealed significant interaction only in the case of high concentrations of quercetin, at least at five orders of magnitude higher than the concentrations needed for cell growth inhibition. All other phenols showed low interactions. 2) Oxygen species production after mitogen stimulation and H²O²2 sensitivity of these cell lines did not correlate with the observed antiproliferative effects, ruling out such a mode of action. 3) NO production revealed two different patterns: LNCaP and DU145 cells produced high concentrations of NO, whereas PC3 cells produced low concentrations. Phorbol ester stimulation of cells did not reveal any additional effect in LNCaP and DU145 cells, whereas it enhanced the secretion of NO in PC3 cells. Polyphenols decreased NO secretion. This effect correlates with their antiproliferative action and the inhibition of inducible NO synthase. It is therefore proposed that the antiproliferative effect of polyphenols is mediated through the modulation of NO production. In conclusion, our data show a direct inhibitory effect of low concentrations of antioxidant wine phenols on the proliferation of human prostate cancer cell lines mediated by the production of NO, further suggesting potential beneficial effects of wine and other phenol-containing foods or drinks for the control of prostate cancer cell growth.

Activated T‐lymphocytes with myelosuppressive properties in patients with chronic idiopathic neutropenia

To characterize the cellular components responsible for the impaired granulopoiesis in chronic idiopathic neutropenia (CIN), we investigated the origin of the proapoptotic cytokine producing cells in the bone marrow (BM) microenvironment of CIN patients. We found that the interferon gamma (IFNγ) and/or Fas‐ligand expressing cells in patient BM mononuclear cells and long‐term BM culture stroma cells were the CD3+ T‐lymphocytes but not the CD14+ monocytes/macrophages. The percentage of activated T‐lymphocytes was increased in patients’ BM as indicated by the proportions of human leucocyte antigen (HLA)‐DR+, CD25+, CD38+, CD69+ and Fas+ cells within the CD3+ fraction. Intracellular IFNγ expression was higher in the BM than peripheral blood of the patients and was associated with increased BM T‐lymphocyte numbers. In crossover experiments, patient CD3+ T‐lymphocytes conferred autologous and allogeneic haemopoietic progenitor cell colony inhibition. Patients’ T‐cell receptor repertoire and polymerase chain reaction analysis did not reveal any clonal T‐lymphocyte expansion, suggesting the absence of a direct, antigen‐driven recognition of CD34+ myeloid progenitor cells by patient T‐lymphocytes. We conclude that CIN patients have increased number of activated T‐lymphocytes in the BM, probably in the setting of a localized polyclonal immune reaction and that these cells confer an inhibitory effect on myelopoiesis through myelosuppressive cytokines including Fas‐ligand and IFNγ.

Impaired megakaryopoiesis in patients with chronic idiopathic neutropenia is associated with increased transforming growth factor β1 production in the bone marrow

Patients with chronic idiopathic neutropenia (CIN) display relatively low peripheral blood platelet counts and hypo‐lobulated megakaryocytes in the bone marrow (BM). The underlying pathogenetic mechanismswere probed by studying the reserves and clonogenic potential of BM megakaryocytic progenitor cells using flow‐cytometry and a collagen‐based clonogenic assay for the identification of megakaryocyte colony‐forming units (CFU‐Meg). Thrombopoietin (TPO) and transforming growth factor‐β1 (TGFβ1) levels were also evaluated in long‐term BM culture supernatants using an enzyme‐linked immunosorbent assay. CIN patients (n = 39) showed a low proportion of BM CD34+/CD61+ megakaryocytic progenitor cells and low frequency of early and mixed CFU‐Meg in the BM mononuclear, but not CD34+, cell fraction, compared with healthy controls (n = 20). TPO and TGFβ1 levels were significantly higher in patients compared with controls. TPO levels inversely correlated with platelet counts whereas TGFβ1 values correlated inversely with CD34+/CD61+ and CFU‐Meg megakaryocytic progenitor cell numbers and positively with TPO levels. The addition of an anti‐TGFβ1 neutralising antibody significantly increased the numbers of CFU‐Meg in CIN patients but not in controls, compared with baseline. These data suggest that increased local production of TGFβ1 probably affects the BM megakaryocytic progenitor cell growth in CIN whereas the compensatory production of TPO finally balances the TGFβ1‐induced inhibitory effect.

Increased cell apoptosis in bone marrow trephine biopsies and immunomagnetically isolated myeloid progenitor cells in patients with chronic idiopathic neutropenia

The frequency of apoptotic cells in bone marrow trephine biopsies and cytospins of immunomagnetically isolated myeloid progenitor cells was determined in 39 patients with chronic idiopathic neutropenia (CIN) and 12 hematologically normal individuals using the in situ end-labeling (ISEL) apoptosis detection method. We found that 66.7% of the patients but none of the normal controls displayed apoptotic cells equal to or higher than 5% of the total mononuclear cells in bone marrow biopsies (p<0.01). In the double stain, we also found that the proportion of apoptotic CD15+ myeloid precursor cells did not differ significantly between patients and control subjects, while the proportion of apoptotic CD34+ hemopoietic cells could not be estimated with accuracy because of the presence of CD34+ endothelial cells. Significantly increased apoptosis was noted in cytospins of immunomagnetically isolated patient CD34+ and CD34+/CD33+ cells but not CD34-/CD33+ cells, compared to the controls (p<0.001, p<0.02 and p>0.05, respectively). These findings confirm and extend our previous observations in flow-cytometric studies of apoptosis in CIN, indicating that increased apoptosis in CIN bone marrow concerns mainly the CD34+ and CD34+/CD33+ progenitor cell compartments. We conclude that the accelerated apoptosis in these compartments may account for the impaired neutrophil production in CIN patients.

Lymphopenia in patients with chronic idiopathic neutropenia is associated with decreased number of T‐lymphocytes containing T‐cell receptor excision circles

Objectives:  Chronic idiopathic neutropenia (CIN) is a disorder of granulopoiesis characterized by the presence of activated T‐lymphocytes that induce/sustain apoptosis of bone marrow (BM) granulocytic progenitors. T‐cell lymphopenia is commonly found in CIN. The aim of the study is to probe the mechanisms underlying T‐cell lymphopenia in CIN.