GE132+Natural: Novel promising dietetic supplement with antiproliferative influence on prostate, colon, and breast cancer cells.

PURPOSE: Natural products have been investigated for promising new leads in pharmaceutical development. The purpose of this study was to analyze the biological effect of GE132+Natural, a novel supplement consisting of 5 compounds: Resveratrol, Ganoderma lucidum, Sulforaphane, Lycopene and Royal jelly. METHODS: The antiproliferative activity of GE132+Natural was tested on 3 different human cancer cell lines: MCF7 (breast cancer cells), PC3 (prostate cancer cells), and SW480 (colon cancer cells), as well as on EA.hy 926 (normal human endothelial cell line). In addition, the cytotoxicity of GE132+- Natural on the proliferation of primary human mesenchymal stem cells isolated from dental pulp (DP=MSC), along with its in vitro impact on different peripheral blood parameters, was determined. RESULTS: The results revealed high antiproliferative activity of GE132+Natural on all tested cancer cell lines (PC3, MCF7 and SW480), as well as on the EA.hy 926 endothelial cell line in a dose-dependent manner. However, applied in a wide range of concentrations GE132+Natural did not affect both the proliferation of primary mesenchymal stem cells and the peripheral blood cells counts. CONCLUSION: The data obtained demonstrated that GE132+Natural is effective in inhibiting cancer cell proliferation, indicating its potential beneficial health effects. In addition, the results pointed that adult mesenchymal stem cells might be valuable as a test system for evaluating the toxicity and efficacy of new medicines or chemicals.

Effects of IL-17 on erythroid progenitors growth: involvement of MAPKs and GATA transcription factors.

Interleukin-17 is Th17 cell cytokine implicated in regulation of hematopoiesis and inflammation. Besides promoting granulopoiesis, we have previously shown that IL-17 also affects erythropoiesis stimulating the development of early erythroid progenitors, BFU-E, but suppressing, at least partly via p38 MAPK, the growth of late stage erythroid progenitors, CFU-E. The aim of the present study was to investigate the involvement of other MAPKs, JNK and ERK1/2, as well as GATA transcription factors, in IL-17-mediated effects on murine bone marrow erythroid progenitors. Data obtained by use of specific MAPKs inhibitors indicated that MEK1/2-ERK1/2 MAPK signaling mediates IL-17-induced CFU-E inhibition, as well as that JNK and/or MEK1/2-ERK1/2 MAPKs activation underlies IL-17-induced stimulation of BFU-E growth. Furthermore, Western blot analyses demonstrated no effect on early hematopoiesis transcription factor, GATA-2, and enhanced expression level of erythroid-specific factor GATA-1 in murine bone marrow cells after IL-17 stimulation, which in light of previous reports that GATA-1 overexpression inhibits erythroid differentiation, could be related to IL-17-mediated inhibition of CFU-E growth. Although, no contribution for p38, JNK and ERK MAPKs in IL-17-induced GATA-1 expression was shown, data obtained using specific inhibitors pointed to the role of JNK and MEK1/2-ERK1/2 in GATA-1 downregulation. Overall, obtained data gave an insight into the mechanisms by which IL-17 exerts its effects on erythropoiesis, implying the involvement of JNK and ERK MAPKs, as well as GATA-1, in IL-17-regulated growth of erythroid progentors.

Combined effect of IL-17 and blockade of nitric oxide biosynthesis on haematopoiesis in mice.

AIM: The study was undertaken to extend our investigation concerning both the in vivo activity of interleukin (IL)-17 and the specific role of nitric oxide (NO) in IL-17-induced effects in the process of haematopoiesis. METHODS: CBA mice were simultaneously treated with IL-17 and/or nitric oxide synthase (NOS) inhibitor, l-NAME, for 5 days and changes within various haematopoietic cell lineages in bone marrow, spleen and peripheral blood were analysed. RESULTS: Findings showed that administration of both IL-17 and l-NAME stimulated increase in net haematopoiesis in normal mice. IL-17-enhanced myelopoiesis was characterized by stimulation of both femoral and splenic haematopoietic progenitor cells and morphologically recognizable granulocytes. Additionally, IL-17 induced alterations in the frequency of erythroid progenitor cells in both bone marrow and spleen, accompanied with their mobilization to the peripheral blood. As a consequence of these changes in the erythroid cell compartments, significant reticulocytosis was observed, which evidenced that in IL-17-treated mice effective erythropoiesis occurred. Exposure of mice to NOS inhibitor also increased the number of both granulocyte-macrophage and erythroid progenitors in bone marrow and spleens, and these alterations were followed by the mobilization of erythroid progenitors and elevated content of reticulocytes in peripheral blood. The specific role of NO in IL-17-induced haematopoiesis was demonstrated only in the IL-17-reducing effect on bone marrow late stage erythroid progenitors, CFU-E. CONCLUSION: The results demonstrated the involvement of both IL-17 and NO in the regulation of haematopoietic cell activity in various haematopoietic compartments. They further suggest that IL-17 effects are differentially mediated depending on the haematopoietic microenvironments.

ERK 1,2 and p38 pathways are involved in the proliferative stimuli mediated by urokinase in osteoblastic SaOS-2 cell line.

Bone metastases from prostate origin generate an osteoblastic reaction that is expressed in vitro by increased osteoblast proliferation. The urokinase-like plasminogen activator (u-PA) present in the media conditioned by tumoral prostatic cells acting as a ligand of the cellular membrane receptor (u-PAR), has been identified as the specific factor that modulates this proliferative reaction. The present study represents an effort to unravel the intracellular pathway by which u-PA activates osteoblastic proliferation and to evaluate the role of cellular receptor u-PAR in this proliferative phenomenon. Our results show that in vitro u-PA stimulates proliferation of SaOS-2 osteoblastic cells by activating the MAP kinase route of ERK 1 and 2 and the p38 pathway. These results are in accordance with the inhibition of intermediate activation and cell proliferation by PD 098059 and SB 203580, specific inhibitors of MEK and p38, respectively. We also show that SaOS-2 cells increase their proliferative response when cells are plated onto vitronectin, the second natural ligand of u-PAR, and that culturing SaOS-2 cells in the presence of u-PA represents a stimuli for u-PAR expression. On the basis of these results we propose that osteoblastic cells respond to the prostate-derived u-PA stimuli in a very efficient manner that includes the utilization of two different signaling routes and the stimulation of the expression of the u-PA receptor.

Genistein and curcumin block TGF-beta 1-induced u-PA expression and migratory and invasive phenotype in mouse epidermal keratinocytes.

Transforming growth factor-beta 1 (TGF-beta 1) stimulates migration/invasion of mouse transformed keratinocytes and increases urokinase (u-PA) expression/secretion. In this report, we analyzed the biological behavior of two naturally occurring inhibitors of protein tyrosine kinases, genistein and curcumin, that could abrogate the enhancement of u-PA levels induced by TGF-beta 1 in transformed keratinocytes. Our results showed that genistein and curcumin blocked this response in a dose-dependent manner and also inhibited the TGF-beta 1-induced synthesis of fibronectin, an early responsive gene to the growth factor. Both compounds also reduced TGF-beta 1-stimulated cell migration and invasiveness. These results suggest that a tyrosine kinase-signaling pathway should be involved in TGF-beta 1-mediated increased malignancy of transformed keratinocytes and that genistein and curcumin could play an important role in inhibiting tumor progression.

Involvement of the Ras/MAPK signaling pathway in the modulation of urokinase production and cellular invasiveness by transforming growth factor-beta(1) in transformed keratinocytes.

Transformed PDV keratinocytes respond to TGF-beta(1) by stimulating cell motility and invasiveness concomitantly to enhancement of the urokinase-type plasminogen activator (uPA) expression/secretion. Depletion of extracellular signal-regulated kinase (ERK1, 2) proteins by treatment of PDV cells with antisense oligonucleotides reduced basal uPA production and abolished stimulation of uPA secreted levels and cell motility by TGF-beta(1). PD098059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase (MEK), decreased TGF-beta(1)-induced uPA mRNA expression, secreted activity in a dose-dependent manner, and abrogated TGF-beta(1)-stimulated cell motility and invasiveness. PDV-derived dominant-negative RasN17 cell transfectants secreted similar amounts of uPA and exhibited similar invasive abilities as the parental cells or control clones, but were unable to respond to TGF-beta(1) for stimulation of uPA-secreted levels and invasiveness. These results suggest that a Ras/MAPK transduction pathway is involved in the invasive response of transformed keratinocytes to TGF-beta(1).

Bone extracellular matrix stimulates invasiveness of estrogen-responsive human mammary MCF-7 cells.

Bone is the most frequent site of metastasis in breast cancer. This causes destructive osteolytic lesions. To achieve metastasis to bone, breast cancer cells must proliferate in a new microenvironment, arrest on extracellular matrix and invade. Breast cancer cells progress in the invasive processes only if they destroy bone with the assistance of osteoclasts. In this work, we present data suggesting that MCF-7 cells, an estradiol receptor-positive cell line that exhibits modest invasive capacity, proliferate in the presence of soluble factors secreted by the osteogenic cell line SaOS-2. The cells acquire a more aggressive phenotype when cultured on an extracellular matrix produced by the same osseous cell line. Acquisition of the invasive phenotype appears to be related to the capacity of bone extracellular matrix to induce the expression of urokinase-like plasminogen activator by MCF-7 cells, which is specific for MCF-7 cells, given that MDA-231 cells, an estradiol receptor-negative and more aggressive cell line, did not show significant changes when cultured in the presence of soluble and insoluble bone factors.

Urokinase expression and binding activity associated with the transforming growth factor beta1-induced migratory and invasive phenotype of mouse epidermal keratinocytes.

Transforming growth factor beta1(TGF-beta1) is a stimulator of malignant progression in mouse skin carcinogenesis. TGF-beta1 exerts a differential effect on cultured nontumorigenic (MCA3D cell line) and transformed (PDV cell line) keratinocytes. Whereas MCA3D cells are growth arrested and committed to die in the presence of the factor, it induces a reversible epithelial-fibroblastic conversion in PDV cells. This conversion is associated in vivo with a squamous-spindle cell carcinoma transition. Here we have investigated the role of urokinase (uPA) during malignant progression of transformed epidermal keratinocytes. We show that the levels of uPA expression/secretion, and the uPA binding activity to the cell surface, correlate with the invasive and malignant potentials of mouse epidermal cell lines. TGF-beta1 enhanced uPA production, the number of uPA cell surface binding sites, and the expression of the plasminogen activator inhibitor PAI-1, in transformed PDV cells, but had no major effect on nontumorigenic MCA3D keratinocytes. Increased uPA production depended on the presence of the factor in the culture medium and occurred concomitantly to the stimulation of the migratory and invasive abilities of PDV cells. Synthetic peptides containing the amino terminal sequence of the mature mouse uPA inhibited the binding of uPA to the cell surface and decreased TGF-beta1-induced cell motility and invasiveness. These results demonstrate that the uPA system mediates at least part of the migratory and invasive phenotype induced by TGF-beta1 in transformed keratinocytes, and suggest a role for uPA on the changes that lead to the appearance of spindle carcinomas.

Effect of desialylated human chorionic gonadotropin (hCG) on the bioactivity of rat Leydig cells.

Human chorionic gonadotropin is a glycoprotein hormone that, like LH, stimulates steroidogenesis in gonadal cells. Using a desialylation process. 95 per cent of the sialic acid residues from an intact standard hCG molecule were eliminated and then the electrophoretic properties and the bioactivity of the desialylated hCG were determined. Using rat Leydig cells as a biological model, the binding affinity to LH receptors of Leydig cell membranes, steroidogenic activity and second messenger production were studied. The results indicate that the loss of sialic acid from the hCG molecule slightly increases the binding activity to LH receptors and results in steroidogenic activity with an increased ED50. Cyclic AMP production was significantly reduced however and arachidonic acid release was not observed. Several possible mechanisms that could explain these results are discussed.

Soluble factors secreted by PC-3 cells induce structural changes in proteoglycans produced by fetal rat osteoblasts.

Metastatic prostate cancer is unique in its ability to induce an osteoblastic reaction in the skeleton, a phenomenon which is followed by impairment of the mineralization process. We have proposed previously that soluble factors present in a medium conditioned by prostatic PC-3 cells (PC-3 CM) induce a rearrangement of bone extracellular matrix (ECM) which precedes the inhibition of mineralization. Interstitial collagen is the ECM component which is most affected by these prostatic factors. In this study, we evaluated the synthesis and molecular characteristics of proteoglycans (PG) derived from fetal rat osteoblasts cultured in the presence of PC-3 CM. These soluble factors induce a decrease (15-20%) in the production of PG. The in vitro produced PG display a decreased mean charge density and an increase in the hydrodynamic size of glycosaminoglycan (GAG) chains. No changes were observed in the size of the core protein or in the type of GAG chains of chondroitin sulfate. From these results, we suggest that fetal rat osteoblasts cultured in the presence of PC-3 CM synthesize PG which generate an ECM unable to support proper mineralization. We speculate that the modification of the ECM offers an advantage for tumor expansion.

Genistein inhibits proliferation and in vitro invasive potential of human prostatic cancer cell lines.

Genistein -a natural flavone compound with antitumor activity- has been proposed as an effective agent to prevent the expression of metastasic capacity in hormone-dependent cancers. The present study represents an effort to assess the efficacy of Genistein in inhibiting the proliferation and expression of the in vitro invasive capacity of tumoral prostatic cells with different invasive potential. In a cell culture system, genistein appeared to be cytotoxic and inhibitory of miaration through a Material barrier to PC-3 cells, the more aggressive invasive cell-line studied. DU-145 and LNCaP cells, which are less invasive than PC-3, are less affected by Genistein both with respect to proliferation rate and inhibition of u-PA and 72 kDa Gelatinase secretion. Measurement of the level of tyrosine-phosphoproteins in the three cell lines studied also showed that PC-3 cells are the most sensitive cells, with a possible molecular target in a membrane-bound protein of 130 kDa.

Soluble factors produced by PC-3 prostate cells decrease collagen content and mineralisation rate in fetal rat osteoblasts in culture.

Approximately 70% of patients with prostate cancer develop bone metastases in the advanced state of the disease. In the present study, we sought to test the hypothesis that prostatic cancer cells produce factors that inhibit the mineralisation process in vitro, decreasing the content of type I collagen in rat fetal calvaria osteoblasts. We investigated the capacity of conditioned media (CM) from the human prostatic tumour cell line PC-3 to inhibit the expression of the differentiation programme on osteoblasts in culture, with a primary focus on type I collagen synthesis and degradation. Our results show that PC-3 CM inhibits collagen synthesis and stimulates the production of interstitial collagenase from osteoblasts. A consequential decrease in the content of immunoreactive type I collagen was observed. We have previously demonstrated that PC-3 CM blocks osteoblast differentiation in culture. We propose that under the effect of factors present in PC-3 CM, osteoblastic cells retain the undifferentiated phenotype.

Prostate-derived soluble factors block osteoblast differentiation in culture.

Bone metastasis is a common event and a major cause of morbidity in prostate cancer patients. After colonization of bone, prostate cells induce an osteoblastic reaction which is not associated with marrow fibrosis (i.e., osteoblast but not fibroblast proliferation). In the present study we test the hypothesis that the tumoral prostatic cell line (PC-3) secretes factors that block the osteoblast differentiation process, resulting in an increase of the relative size of the proliferative cell pool. Our results, using fetal rat calvaria cells in culture, show that conditioned medium from PC-3 cells (PC-3 CM) stimulates osteoblast proliferation and inhibits both alkaline phosphatase (AP) activity (an early differentiation marker) and the mineralization process, measured as calcium accumulation (late differentiation marker). The inhibition of the expression of AP and mineralization depends on the presence of PC-3 CM during the proliferative phase of culture and suggests that both processes occur in a nonsimultaneous fashion. The inhibitory effect of PC-3 CM was not reverted by dexamethasone, which would indicate that prostatic-derived factors and the glucocorticoid do not share a common site of action. Measurement of the proliferative capacity of subcultures from control and treated cells demonstrates that PC-3 CM treatment induces the maintenance of the proliferative potential that characterizes undifferentiated precursor cells.

The secretion of urokinase-like plasminogen activator is inhibited by microtubule-interacting drugs.

The secretion of proteinases into the extracellular matrix is one of the main features of tumour cells, as related to their invasive behaviour. Considering the role of the microtubule cytoskeleton, and particularly the action of microtubule-associated protein (MAPs) in mediating protein secretion, the effects of the anti-microtubule drugs estramustine and taxol, on the secretion of urokinase-type plasminogen activator (u-PA) and the 72 kDa gelatinase were investigated. Treatment of 5637 bladder carcinoma cells with estramustine and taxol inhibited u-PA secretion into the conditioned medium in a drug concentration-dependent fashion. This inhibition was confirmed by determinations of u-PA enzymatic activities and by measurements of the levels of immunoreactive activator. Studies using gelatin zymograms also showed an inhibition of another tumoural proteinase namely the 72 kDa gelatinase. Time-course uptake experiments showed that estramustine was incorporated into the cells, a process which depended on temperature. On the other hand, immunofluorescence studies indicated that the microtubule network was affected by taxol with the formation of bundles of microtubules at different cell domains. Minor effects were visualized after treatment of the cells with estramustine-phosphate, a drug that blocks primarily the action of microtubule-associated proteins. The studies provide a way to analyse the relationships between u-PA secretion and the integrity of the cytoskeletal network.

Surface-associated plasminogen activation in leukemic cells: interaction with extracellular matrix.

In WEHI-3B murine leukemic cells, plasminogen activator and plasminogen binding sites are associated with the cell membrane. The putative receptor for the zymogen exhibits low affinity for the ligand (dissociation constant of 0.38 microM and a high binding capacity (40,000 sites per cell). Plasminogen also binds in a cooperative fashion to type I collagen with an affinity which is higher than that displayed by cells. Collagen-bound plasminogen can be activated by cells preincubated with plasminogen in a manner that cells develop the capacity to adhere to type I collagen. The activation of collagen-bound plasminogen by cellular urokinase-like plasminogen activator (u-PA) was 60% more efficient than the activation of the soluble (not bound) form of plasminogen. These results suggest that in the invasive phenomena, WEHI cells operate as carriers of plasminogen from plasma to tissue. In addition, collagen can serve as a reservoir of zymogen in the extracellular matrix milieu through direct binding to plasminogen and at the same time allow more efficient plasminogen activation.

Membrane-associated procollagenase of leukemic cells is activated by urokinase-type plasminogen activator.

Murine leukemic cells from the WEHI-3B line, present in the cell surface a latent collagenase activity which is activated proteolytically. In this paper we show that this enzyme is activated by plasmin generated by the activity of a urokinase-like plasminogen activator (u-PA) also present in the surface of these cells. Using a reverse fibrin autography method we found that u-PA is the major proteolytic activity present in the cell membranes. This fact suggests that u-PA could represent a normal activating system for this collagenase.

Extracellular calcium modulates proliferation of factor dependent hemopoietic cells.

Blockade of the calcium channel inhibited, in a dose-dependent manner, the proliferation of the IL-3 dependent FDCP-1 cell line and normal murine bone marrow cells. Similar results were obtained by lowering the amount of extracellular calcium using specific chelators or a calcium-free medium. These results suggest that factor-dependent cell proliferation is highly sensitive to fluctuations in the concentration of extracellular calcium.

The antineoplastic agent estramustine and the derivative estramustine-phosphate inhibit secretion of interleukin-3 in leukemic cells. Possible roles of MAPs.

The antineoplastic drug estramustine is an adduct of estradiol and nor-nitrogen mustard. It has been shown that this drug interferes with microtubule assembly, an effect mediated by estramustine interaction with microtubule-associated proteins (MAPs). In the present report we demonstrate that estramustine and the phosphorylated derivative of the drug, estramustine-phosphate, inhibit the secretion of interleukin-3 by WEHI-3B cells. These studies also show that the estramustine derivative specifically interacts with a MAPs component found in these cells, which exhibited characteristics ressembling those of tau protein isoforms. Western blots using a unique monoclonal antibody MTB6.22 that recognizes microtubule-binding domains on MAPs, indicated that this WEHI protein factor contained the antigenic determinant that are functionally significant for microtubule assembly. ELISA assays using this antibody, also showed a decrease in the levels of the immunoreactive protein in WEHI cells after treatment with EMP. Interestingly, it has been recently described that the action of estramustine-phosphate is mediated by a direct interaction with MAP-binding sites on the microtubule surface, which are recognized by the site-specific monoclonal antibody. These findings together with immuno-precipitation experiments using anti-interleukin-3 antibodies and the inhibitory effect of the estramustine derivative on WEHI secretion process suggest that this anti-mitotic agent may block IL-3 secretion by a mechanism involving its interaction with a 'tau-like' MAPs component present in these cells.

Binding and production of insulin-like growth factor-I in rat mammary gland.

1. Mammary tissue from pregnant rat presents low and high affinity IGF-I functional receptors. 2. Mammary explants from pregnant and lactating rats secrete IGF-I and its production was related to the developmental stage of the gland. 3. An inverse relationship between IGF-I production and tissue binding capacity was observed.