Athina Damianaki

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.

Opioid alkaloids and casomorphin peptides decrease the proliferation of prostatic cancer cell lines (LNCaP, PC3 and DU145) through a partial interaction with opioid receptors

Opioid agonists (ethylketocyclazocine, etorphine, [D-Ala2,D-Leu5]enkephalin (DADLE), [D-Ala2, N-Me-Phe4-Gly-ol]enkephalin (DAGO), [D-Ser2,Leu5]enkephalin-Thr6 (DSLET) and morphine were found to inhibit the proliferation of human prostate cancer cell lines (LNCaP, DU145, and PC3), in a dose-dependent manner. The 50% inhibitory concentrations (IC50) were in the picomolar range. In many cases, this effect was antagonized by the general opioid antagonist, diprenorphine, indicating the existence of specific opioid binding sites. Saturation binding experiments with selective ligands and effectors showed no opioid sites on the LNCaP cell line, kappa1 and mu sites on the PC3 cell line, and kappa1, kappa3 and mu sites on the DU145 cell line. In other cases, the opioid effect was not antagonized by diprenorphine, indicating that the action of opioids might be mediated through other membrane receptors. Furthermore, casomorphin peptides, issued from bovine alpha- (alpha-casein-90-95 and alpha-casein-90-96) and beta-caseins (beta-casomorphin and beta-casomorphin-1-5), and human alphaS1-casein (alphas -casomorphin and alphaS1-casomorphin amide) inhibited cell proliferation of human prostate cell lines, also by a mechanism partly involving opioid receptors. As opioid neurons can be found in the prostate gland, and casomorphin peptides might reach the gland through the general circulation, the above findings indicate a putative role of opioids in 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.

Normal bone marrow hematopoietic stem cell reserves and normal stromal cell function support the use of autologous stem cell transplantation in patients with multiple sclerosis.

Summary:Bone marrow (BM) stem cell reserves and function and stromal cell hematopoiesis supporting capacity were evaluated in 15 patients with multiple sclerosis (MS) and 61 normal controls using flow cytometry, clonogenic assays, long-term BM cultures (LTBMCs) and enzyme-linked immunosorbent assays. MS patients displayed normal CD34+ cell numbers but a low frequency of colony-forming cells (CFCs) in both BM mononuclear and purified CD34+ cell fractions, compared to controls. Patients had increased proportions of activated BM CD3+/HLA-DR+ and CD3+/CD38+ T cells that correlated inversely with CFC numbers. Patient BM CD3+ T cells inhibited colony formation by normal CD34+ cells and patient CFC numbers increased significantly following immunomagnetic removal of T cells from BMMCs, suggesting that activated T cells may be involved in the defective clonogenic potential of hematopoietic progenitors. Patient BM stromal cells displayed normal hematopoiesis supporting capacity indicated by the CFC number in the nonadherent cell fraction of LTBMCs recharged with normal CD34+ cells. Culture supernatants displayed normal stromal derived factor-1 and stem cell factor/kit ligand but increased flt-3 ligand levels. These findings provide support for the use of autologous stem cell transplantation in MS patients. The low clonogenic potential of BM hematopoietic progenitors probably reflects the presence of activated T cells rather than an intrinsic defect.

Investigation of bone marrow mesenchymal stem cells (BM MSCs) involvement in idiopathic pulmonary fibrosis (IPF)

BACKGROUND Experimental data have provided evidence that progenitor cells of bone marrow (BM) origin may play a role in the fibrogenic process of the lung. OBJECTIVE To probe the possible involvement of BM mesenchymal stem cells (MSCs) in the pathophysiology of Idiopathic Pulmonary Fibrosis (IPF) by investigating the molecular profile of these cells. DESIGN BM MSCs were studied in 10 IPF patients and 10 healthy controls. MSCs were identified by their immunophenotypic characteristics and their potential to differentiate towards adipocytes/osteocytes/chondrocytes. We evaluated the mRNA expression of genes involved in the lung injury of IPF, namely the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), transforming growth factor beta-1 (TGF-beta1) and the axis stromal-cell-derived factor-1 (SDF-1)/CXCR4 in BM MSCs using quantitative RT-PCR. RESULTS The BM MSCs of IPF patients displayed normal immunophenotypic characteristics and differentiation potential. No statistically significant difference was found between patients and controls in VEGF and FGF mRNA expression. TGF-beta1 was not expressed in either patients or controls. A significant increase in SDF-1-TR1 and CXCR4 mRNA expression was detected in IPF patients (1.6 x 10(25) +/- 1.2 x 10(25) and 3.1 x 10(7) +/- 3.1 x 10(7), respectively) compared to controls (0.32 x 10(25) +/- 0.07 x 10(25) and 1.67 x 10(7) +/- 0.30 x 10(7), respectively) (p = 0.001 and p = 0.001, respectively) whereas SDF-1 levels in MSC supernatants were similar in patients and controls. CONCLUSIONS The increased CXCR4 expression by patient MSCs suggests that the BM is probably implicated in the pathophysiology of IPF by mobilizing MSCs in response to or preceding lung injury. The potential role of BM MSCs in IPF is another interesting field for further investigation.

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.

Mesenchymal stem cells contribute to the abnormal bone marrow microenvironment in patients with chronic idiopathic neutropenia by overproduction of transforming growth factor-β1.

Chronic idiopathic neutropenia (CIN) is a granulopoiesis disorder associated with an inhibitory bone marrow (BM) microenvironment consisting of activated T-lymphocytes and pro-inflammatory mediators. In this study, we investigated the possible involvement of BM mesenchymal stem cells (MSCs) in the pathophysiology of CIN by assessing the frequency and function of BM MSCs in terms of the proliferative/clonogenic characteristics, the differentiation capacity, the potential to produce pro-inflammatory cytokines, and the ability to suppress T-cell proliferation. The frequency, differentiation capacity toward adipocytes, chondrocytes, or osteoblasts, and immunosuppressive potential to inhibit mitogen-induced T-cell proliferation did not differ significantly between patient (n = 14) and normal (n = 21) MSCs. Tumor necrosis factor-α, interleukin-1β, and interleukin-6 levels in MSC supernatants did not differ significantly between patients and controls; however, transforming growth factor (TGF)-β1 levels were significantly elevated in patients, particularly in those displaying the -509C/T TGF-β1 polymorphism. Patient MSCs displayed defective proliferative/clonogenic potential, which could not be attributed to altered cellular survival characteristics or to increased TGF-β1 production as TGF-β1 neutralization did not restore the impaired colony formation by patient MSCs. We conclude that although BM MSCs do not exert a significant role in the immune deregulation associated with CIN, they contribute to the inhibitory microenvironment by overproducing TGF-β1, at least in patients displaying the -509C/T polymorphism.

Differential expression of cell cycle and WNT pathway-related genes accounts for differences in the growth and differentiation potential of Wharton’s jelly and bone marrow-derived mesenchymal stem cells

BackgroundIn view of the current interest in exploring the clinical use of mesenchymal stem cells (MSCs) from different sources, we performed a side-by-side comparison of the biological properties of MSCs isolated from the Wharton’s jelly (WJ), the most abundant MSC source in umbilical cord, with bone marrow (BM)-MSCs, the most extensively studied MSC population.MethodsMSCs were isolated and expanded from BM aspirates of hematologically healthy donors (n = 18) and from the WJ of full-term neonates (n = 18). We evaluated, in parallel experiments, the MSC immunophenotypic, survival and senescence characteristics as well as their proliferative potential and cell cycle distribution. We also assessed the expression of genes associated with the WNT- and cell cycle-signaling pathway and we performed karyotypic analysis through passages to evaluate the MSC genomic stability. The hematopoiesis-supporting capacity of MSCs from both sources was investigated by evaluating the clonogenic cells in the non-adherent fraction of MSC co-cultures with BM or umbilical cord blood-derived CD34+ cells and by measuring the hematopoietic cytokines levels in MSC culture supernatants. Finally, we evaluated the ability of MSCs to differentiate into adipocytes and osteocytes and the effect of the WNT-associated molecules WISP-1 and sFRP4 on the differentiation potential of WJ-MSCs.ResultsBoth ex vivo-expanded MSC populations showed similar morphologic, immunophenotypic, survival and senescence characteristics and acquired genomic alterations at low frequency during passages. WJ-MSCs exhibited higher proliferative potential, possibly due to upregulation of genes that stimulate cell proliferation along with downregulation of genes related to cell cycle inhibition. WJ-MSCs displayed inferior lineage priming and differentiation capacity toward osteocytes and adipocytes, compared to BM-MSCs. This finding was associated with differential expression of molecules related to WNT signaling, including WISP1 and sFRP4, the respective role of which in the differentiation potential of WJ-MSCs was specifically investigated. Interestingly, treatment of WJ-MSCs with recombinant human WISP1 or sFRP4 resulted in induction of osteogenesis and adipogenesis, respectively. WJ-MSCs exhibited inferior hematopoiesis-supporting potential probably due to reduced production of stromal cell-Derived Factor-1α, compared to BM-MSCs.ConclusionsOverall, these data are anticipated to contribute to the better characterization of WJ-MSCs and BM-MSCs for potential clinical applications.

Pro‐inflammatory bone marrow milieu in patients with chronic idiopathic neutropenia is associated with impaired local production of interleukin‐10

The levels of interleukin‐10 (IL‐10) were evaluated in long‐term bone marrow (BM) culture supernatants from 54 patients with chronic idiopathic neutropenia (CIN) and 30 healthy volunteers using enzyme‐linked immunoabsorbent assay. Cytokine levels were significantly reduced in patients, compared with controls, and strongly correlated with peripheral blood neutrophil counts. Low levels of supernatant IL‐10 were associated with increased values of supernatant IL‐1β, tumour necrosis factor‐α, IL‐6 and transforming growth factor‐β1. We suggest that the pro‐inflammatory milieu in the BM of CIN patients may be causatively related to the impaired production of IL‐10, a cytokine normally displaying strong anti‐inflammatory properties.