Tumor necrosis factor alpha triggers proliferation of adult neural stem cells via IKK/NF-kappaB signaling

BACKGROUND: Brain inflammation has been recognized as a complex phenomenon with numerous related aspects. In addition to the very well-described neurodegenerative effect of inflammation, several studies suggest that inflammatory signals exert a potentially positive influence on neural stem cell proliferation, migration and differentiation. Tumor necrosis factor alpha (TNF-alpha) is one of the best-characterized mediators of inflammation. To date, conclusions about the action of TNF on neural stem or progenitor cells (NSCs, NPCs) have been conflicting. TNF seems to activate NSC proliferation and to inhibit their differentiation into NPCs. The purpose of the present study was to analyze the molecular signal transduction mechanisms induced by TNF and resulting in NSC proliferation. RESULTS: Here we describe for the first time the TNF-mediated signal transduction cascade in neural stem cells (NSCs) that results in increased proliferation. Moreover, we demonstrate IKK-alpha/beta-dependent proliferation and markedly up-regulated cyclin D1 expression after TNF treatment. The significant increase in proliferation in TNF-treated cells was indicated by increased neurosphere volume, increased bromodeoxyuridin (BrdU) incorporation and a higher total cell number. Furthermore, TNF strongly activated nuclear factor-kappa B (NF-kappaB) as measured by reporter gene assays and by an activity-specific antibody. Proliferation of control and TNF-treated NSCs was strongly inhibited by expression of the NF-kappaB super-repressor IkappaB-AA1. Pharmacological blockade of IkappaB ubiquitin ligase activity led to comparable decreases in NF-kappaB activity and proliferation. In addition, IKK-beta gene product knock-down via siRNA led to diminished NF-kappaB activity, attenuated cyclin D1 expression and finally decreased proliferation. In contrast, TGFbeta-activated kinase 1 (TAK-1) is partially dispensable for TNF-mediated and endogenous proliferation. Understanding stem cell proliferation is crucial for future regenerative and anti-tumor medicine. CONCLUSION: TNF-mediated activation of IKK-beta resulted in activation of NF-kappaB and was followed by up-regulation of the bona-fide target gene cyclin D1. Activation of the canonical NF-kappaB pathway resulted in strongly increased proliferation of NSCs.

Glucose metabolism by lymphocytes, macrophages, and tumor cells from Walker 256 tumor-bearing rats supplemented with fish oil for one generation

Here we investigated the effect of lifelong supplementation of the diet with coconut fat (CO, rich in saturated fatty acids) or fish oil (170, rich in n-3 polyunsaturated fatty acids) on tumor growth and lactate production from glucose in Walker 256 tumor cells, peritoneal macrophages, spleen, and gut-associated lymphocytes. Female Wistar rats were supplemented with CO or FO prior to mating and then throughout pregnancy and gestation and then the male offspring were supplemented from weaning until 90 days of age. Then they were inoculated subcutaneously with Walker 256 tumor cells. Tumor weight at 14 days in control rats (those fed standard chow) and CO supplemented was approximately 30 g. Supplementation of the diet with FO significantly reduced tumor growth by 76%. Lactate production (nmol h(-1) mg(-1) protein) from glucose by Walker 256 cells in the group fed regular chow (W) was 381.8 +/- 14.9. Supplementation with coconut fat (WCO) caused a significant reduction in lactate production by 1.6-fold and with fish oil (WFO) by 3.8-fold. Spleen lymphocytes obtained from W and WCO groups had markedly increased lactate production (553 +/- 70 and 635 +/- 150) when compared to non-tumor-bearing rats (similar to 260 +/- 30). FO supplementation reduced significantly the lactate production (297 +/- 50). Gut-associated lymphocytes obtained from W and WCO groups increased lactate production markedly (280 +/- 31 and 276 +/- 25) when compared to non-tumor-bearing rats (similar to 90 +/- 18). FO supplementation reduced significantly the lactate production (168 +/- 14). Lactate production by peritoneal macrophages was increased by tumor burden but there was no difference between the groups fed the various diets. Lifelong consumption of FO protects against tumor growth and modifies glucose metabolism in Walker tumor cells and lymphocytes but not in macrophages. Copyright (C) 2008 John Wiley & Sons, Ltd.

Expression of a dendritic cell maturation marker CD83 on tumor cells from lung cancer patients and several human tumor cell lines: is there a biological meaning behind it?

The present paper shows, for the first time, the membrane expression of the dendritic cell maturation marker CD83 on tumor cells from lung cancer patients. CD83 was also detected on freshly cultured fibroblast-like cells from these tissues and on several adherent human tumor cell lines (lung adenocarcinomas P9, A459 and A549, melanomas A375 and C81-61, breast adenocarcinomas SKBR-3 and MCF-7 and colon carcinoma AR42-J), but not in the non-adherent MOT leukemia cell line. CD83 may have immunosuppressive properties and its expression by cancer cells could have a role in facilitating tumor growth.

High frequency of immature dendritic cells and altered in situ production of interleukin-4 and tumor necrosis factor-alpha in lung cancer

Introduction Antigen-presenting cells, like dendritic cells (DCs) and macrophages, play a significant role in the induction of an immune response and an imbalance in the proportion of macrophages, immature and mature DCs within the tumor could affect significantly the immune response to cancer. DCs and macrophages can differentiate from monocytes, depending on the milieu, where cytokines, like interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) induce DC differentiation and tumor necrosis factor (TNF)-alpha induce DC maturation. Thus, the aim of this work was to analyze by immunohistochemistry the presence of DCs (S100+ or CD1a+), macrophages (CD68+), IL-4 and TNF-alpha within the microenvironment of primary lung carcinomas. Results Higher frequencies of both immature DCs and macrophages were detected in the tumor-affected lung, when compared to the non-affected lung. Also, TNF-alpha-positive cells were more frequent, while IL-4-positive cells were less frequent in neoplastic tissues. This decreased frequency of mature DCs within the tumor was further confirmed by the lower frequency of CD14-CD80+ cells in cell suspensions obtained from the same lung tissues analyzed by flow cytometry. Conclusion These data are discussed and interpreted as the result of an environment that does not oppose monocyte differentiation into DCs, but that could impair DC maturation, thus affecting the induction of effective immune responses against the tumor.

Glypican-3 regulates migration, adhesion and actin cytoskeleton organization in mammary tumor cells through Wnt signaling modulation

Glypican-3 (GPC3) is a proteoglycan involved in migration, proliferation and cell survival modulation in several tissues. There are many reports demonstrating a downregulation of GPC3 expression in some human tumors, including mesothelioma, ovarian and breast cancer. Previously, we determined that GPC3 reexpression in the murine mammary adenocarcinoma LM3 cells induced an impairment of their in vivo invasive and metastatic capacities together with a higher susceptibility to in vitro apoptosis. Currently, the signaling mechanism of GPC3 is not clear. First, it was speculated that GPC3 regulates the insulin-like growth factor (IGF) signaling system. This hypothesis, however, has been strongly challenged. Recently, several reports indicated that at least in some cell types GPC3 serves as a selective regulator of Wnt signaling. Here we provide new data demonstrating that GPC3 regulates Wnt pathway in the metastatic adenocarcinoma mammary LM3 cell line. We found that GPC3 is able to inhibit canonical Wnt signals involved in cell proliferation and survival, as well as it is able to activate non canonical pathway, which directs cell morphology and migration. This is the first report indicating that breast tumor cell malignant properties can be reverted, at least in part, by GPC3 modulation of Wnt signaling. Our results are consistent with the potential role of GPC3 as a metastasis suppressor.

MicroRNA-100 Acts as a Tumor Suppressor in Human Bladder Carcinoma 5637 Cells

Bladder carcinoma is one of the most common tumors in the world and, despite the therapy currently available, most of the patients relapse. Better understanding of the factors involved in disease pathogenesis would provide insights for the development of more effective strategies in treatment. Recently, differential miRNA expression profiles in bladder urothelial carcinomas identified miR-100 down-regulation and miR-708 up-regulation among the most common alterations, although the possible influence of these miRNAs in the control of basic mechanisms in bladder tumors has not been addressed. In this context, the present study aimed to evaluate the in vitro effects of miR-100 forced expression and miR-708 inhibition in the bladder carcinoma cell line 5637. Our results showed that overexpression of miR-100 significantly inhibited growth when compared to controls at both times tested (72 and 96 hours, p<0.01) with a maximum effect at 72 hours reducing proliferation in 29.6 %. Conversely, no effects on cell growth were observed after inhibition of miR-708. MiR-100 also reduced colony formation capacity of 5637 cells by 24.4%. No alterations in cell cycle progression or apoptosis induction were observed. The effects of miR-100 on growth and clonogenicity capacity in 5637 cells evince a possible role of this miRNA in bladder carcinoma pathogenesis. Further studies are necessary to corroborate our findings and examine the potential use of this microRNA in future therapeutic interventions.

Annexin 1: differential expression in tumor and mast cells in human larynx cancer

Annexin 1 protein (ANXA1) expression was evaluated in tumor and mast cells in human larynx cancer and control epithelium. The effect of the exogenous ANXA1 (peptide Ac 2-26) was also examined during the cellular growth of the Hep-2 human larynx epidermoid carcinoma cell line. This peptide inhibited the proliferation of the Hep-2 cells within 144 hr. In surgical tissue specimens from 20 patients with larynx cancer, ultrastructural immunocytochemistry analysis showed in vivo down-regulation of ANXA1 expression in the tumor and increased in mast cells and Hep-2 cells treated with peptide Ac2-26. Combined in vivo and in vitro analysis demonstrated that ANXA1 plays a regulatory role in laryngeal cancer cell growth. We believe that a better understanding of the regulatory mechanisms of ANXA1 in tumor and mast cells may lead to future biological targets for the therapeutic intervention of human larynx cancer. (c) 2007 Wiley-Liss, Inc.

Differential regulation of the release of tumor necrosis factor-alpha and of eicosanoids by mast cells in rat airways after antigen challenge

Background: Rat trachea display a differential topographical distribution of connective tissue mast cells (CTMC) and mucosal mast cells (MMC) that may imply regional differences in the release of allergic mediators such as tumor necrosis factor-alpha (TNF-alpha) and eicosanoids.Aim: To evaluate the role of CTMC and MMC for release of TNF-alpha and eicosanoids after allergenic challenge in distinct segments of rat trachea.Materials and methods: Proximal trachea ( PT) and distal trachea (DT) from ovalbumin (OVA)-sensitized rats, treated or not with compound 48/80 ( 48/80) or dexamethasone, were incubated in culture medium. After OVA challenge, aliquots were collected to study release of TNF-alpha and eicosanoids.Results: Release of TNF-alpha by PT upon OVA challenge peaked at 90 min and decayed at 6 and 24 h. Release from DT peaked at 30-90 min and decayed 6 and 24 h later. When CTMC were depleted with 48/80, OVA challenge exacerbated the TNF-alpha release by PT at all time intervals, while DT exacerbated TNF-alpha levels 6 and 24 h later only. Dexamethasone reduced TNF-alpha production after 90 min of OVA challenge in PT and at 3 and 6h in DT. OVA challenge increased prostaglandin D-2 in DT and leukotriene B-4 in both segments but did not modify prostaglandin E-2 and leukotriene C-4 release.Conclusion: OVA challenge induces TNF-alpha release from MMC, which is negatively regulated by CTMC. The profile of TNF-alpha and eicosanoids depends on the time after OVA challenge and of the tracheal segment considered.

Secretion of tumor necrosis factor-alpha by mouse peritoneal macrophages in the presence of dental sealers, sealapex and endomethasone

After filling root canals, the healing process depends on the chemical composition or physical-chemical properties of the material used, among other factors. All root canal sealers, whether solid or plastic, are foreign matter for the body if they remain in permanent contact with apical and periapical tissues. As a result, the first organic reaction that occurs is an attempt to phagocytize the material. During phagocytosis, macrophages release a large number of cell mediators into the area, among which are cytokines that are essential in intercellular communication and in many physiological and pathophysiological processes. One of these cytokines is tumor necrosis factor-alfa (TNF-α), which acts through links to specific receptors on the cell membrane initiating a cascade of events leading to induction, activation, or inhibition of numerous cytokine-regulated genes in the cell nucleus. The release of TNF-α in a cell culture of mouse peritoneal macrophages incubated with three concentrations (25, 50, and 100 mg/ml) of two endodontic sealers was measured. The solutions containing the calcium hydroxide-based root canal sealer (Sealapex) released fewer units of TNF-α than solutions containing the zinc oxide and eugenol-based sealer (Endomethasone).

Myotoxic phospholipases A2 isolated from Bothrops brazili snake venom and synthetic peptides derived from their C-terminal region: Cytotoxic effect on microorganism and tumor cells

This paper reports the purification and biochemical/pharmacological characterization of two myotoxic phospholipases A2 (PLA2s) from Bothrops brazili venom, a native snake from Brazil. Both myotoxins (MTX-I and II) were purified by a single chromatographic step on a CM-Sepharose ion-exchange column up to a high purity level, showing Mr ∼ 14,000 for the monomer and 28,000 Da for the dimer. The N-terminal and internal peptide amino acid sequences showed similarity with other myotoxic PLA2s from snake venoms, MTX-I belonging to Asp49 PLA2 class, enzymatically active, and MTX-II to Lys49 PLA2s, catalytically inactive. Treatment of MTX-I with BPB and EDTA reduced drastically its PLA2 and anticoagulant activities, corroborating the importance of residue His48 and Ca2+ ions for the enzymatic catalysis. Both PLA2s induced myotoxic activity and dose-time dependent edema similar to other isolated snake venom toxins from Bothrops and Crotalus genus. The results also demonstrated that MTXs and cationic synthetic peptides derived from their 115-129 C-terminal region displayed cytotoxic activity on human T-cell leukemia (JURKAT) lines and microbicidal effects against Escherichia coli, Candida albicans and Leishmania sp. Thus, these PLA2 proteins and C-terminal synthetic peptides present multifunctional properties that might be of interest in the development of therapeutic strategies against parasites, bacteria and cancer. © 2008 Elsevier Inc. All rights reserved.

Detection of cytokeratins in ghost cells of calcifying cystic odontogenic tumor indicates an altered keratinization and hair follicle differentiation for their development

Calcifying cystic odontogenic tumors (CCOTs) are benign cystic lesions of odontogenic origin characterized by an ameloblastoma-like epithelium and the presence of a group of cells named ghost cells. The pattern of cytokeratin (Ck) expression on these lesions remains unclear and needs to be clarified. To this end, the expression of Ck6, Ck13, Ck14, Ck18, and Ck19 in the epithelium lining of 7 cases of CCOTs was evaluated by immunohistochemistry. For this, the epithelium lining was divided into 3 distinct regions: basal layer, suprabasal layer, and the compartment composed of ghost cells. In this study, 6 cases (85.7%) were classified as type 1 and 1 (14.3%) as type 4. All cases were negative for Ck13 and Ck18, despite the epithelial layer, as well as in the ghost cells. Ck6 was only positive in the ghost cells. Positivity for Ck14 and Ck19 was found in the basal and suprabasal layers, including the ghost cells. The results showing positivity for Ck14 and Ck19 in all of the analyzed cases reinforce CCOT as being of odontogenic origin, and the restricted expression of Ck6 in the ghost cells may be indicative that these cells suffer an altered differentiation into hair follicles in CCOTs. © 2013 Elsevier Inc. All rights reserved.

Cytoxicity and Apoptotic Mechanism of Ruthenium(II) Amino Acid Complexes in Sarcoma-180 Tumor Cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)