IKK{gamma} protein is a target of BAG3 regulatory activity in human tumor growth.

BAG3, a member of the BAG family of heat shock protein (HSP) 70 cochaperones, is expressed in response to stressful stimuli in a number of normal cell types and constitutively in a variety of tumors, including pancreas carcinomas, lymphocytic and myeloblastic leukemias, and thyroid carcinomas. Down-regulation of BAG3 results in cell death, but the underlying molecular mechanisms are still elusive. Here, we investigated the molecular mechanism of BAG3-dependent survival in human osteosarcoma (SAOS-2) and melanoma (M14) cells. We show that bag3 overexpression in tumors promotes survival through the NF-kappaB pathway. Indeed, we demonstrate that BAG3 alters the interaction between HSP70 and IKKgamma, increasing availability of IKKgamma and protecting it from proteasome-dependent degradation; this, in turn, results in increased NF-kappaB activity and survival. These results identify bag3 as a potential target for anticancer therapies in those tumors in which this gene is constitutively expressed. As a proof of principle, we show that treatment of a mouse xenograft tumor model with bag3siRNA-adenovirus that down-regulates bag3 results in reduced tumor growth and increased animal survival.

Copper-binding activity of Trefoil factor 1 (TFF1): a new perspective in the study of the multifunctional roles of TFFs.

Trefoil factors (TFFs) are gastrointestinal peptides playing an essential role in the epithelial restitution. Among the three known TFF peptides, TFF1 is characterized by three disulfide bonds producing a compact globular structure and an extended and disordered tail formed by amino- and carboxy-termini. The presence of a cysteine surrounded by several negatively charged residues in this region of the protein, highly conserved in different species, suggests the possible formation of a metal-binding site. Affinity chromatography and mass spectrometric analyses allowed us to demonstrate a selective binding affinity of TFF1 for copper. The binding induces conformational changes in the tertiary structure as demonstrated by circular dichroism experiments, while limited proteolysis revealed an altered access to the cleavage sites in the amino- and carboxy-termini. The results of this study reveal a new property of TFF1 and suggest that copper could influence its biological activities by interfering with the dimerization of the peptide and/or the interaction with mucins or putative TFF receptors.

Evidence for BAG3 modulation of HIV-1 gene transcription.

A family of co-chaperone proteins that share the Bcl-2-associated athanogene (BAG) domain are involved in a number of cellular processes, including proliferation and apoptosis. Among these proteins, BAG3 has received increased attention due to its high levels in several disease models and ability to associate with Hsp70 and a number of other molecular partners. BAG3 expression is stimulated during cell response to stressful conditions, such as exposure to high temperature, heavy metals, and certain drugs. Here, we demonstrate that BAG3 expression is elevated upon HIV-1 infection of human lymphocytes and fetal microglial cells. Furthermore, BAG3 protein was detectable in the cytoplasm of reactive astrocytes in HIV-1-associated encephalopathy biopsies, suggesting that induction of BAG3 is part of the host cell response to viral infection. To assess the impact of BAG3 upregulation on HIV-1 gene expression, we performed transcription assays and demonstrated that BAG3 can suppress transcription of the HIV-1 long terminal repeat (LTR) in microglial cells. This activity was mapped to the kappaB motif of the HIV-1 LTR. Results from in vitro and in vivo binding assays revealed that BAG3 suppresses interaction of the p65 subunit of NF-kappaB with the kappaB DNA motif of the LTR. Results from binding and transcriptional assay identified the C-terminus of BAG3 as a potential domain involved in the observed inhibitory effect of BAG3 on p65 activity. These observations reveal a previously unrecognized cell response, that is, an increase in BAG3, elicited by HIV-1 infection, and may provide a new avenue for the suppression of HIV-1 gene expression.

The antiapoptotic protein BAG3 is expressed in thyroid carcinomas and modulates apoptosis mediated by tumor necrosis factor-related apoptosis-inducing ligand.

CONTEXT: We previously showed that BAG3 protein, a member of the BAG (Bcl-2-associated athanogene) co-chaperone family, modulates apoptosis in human leukemias. The expression of BAG3 in other tumor types has not been extensively investigated so far. OBJECTIVE: The objective of this study was to analyze BAG3 expression in thyroid neoplastic cells and investigate its influence in cell apoptotic response to TNF-related apoptosis-inducing ligand (TRAIL). DESIGN, SETTING, AND PATIENTS: We investigated BAG3 expression in human thyroid carcinoma cell lines, including NPA, and the effect of BAG3-specific small interfering RNA on TRAIL-induced apoptosis in NPA cells. Subsequently, we analyzed BAG3 expression in 30 benign lesions and 56 carcinomas from patients of the Naples Tumor Institute Fondazione Senatore Pascale. MAIN OUTCOME MEASURES: The main outcome measures were: analysis of BAG3 protein in NPA cells by Western blot and immunocytochemistry; analysis of apoptosis in TRAIL-stimulated NPA cells by flow cytometry; and evaluation of BAG3 expression in specimens from thyroid lesions by immunohistochemistry. RESULTS: BAG3 was expressed in human thyroid carcinoma cell lines; small interfering RNA-mediated downmodulation of its levels significantly (P < 0.0195) enhanced NPA cell apoptotic response to TRAIL. The protein was not detectable in 19 of 20 specimens of normal thyroid or goiters, whereas 54 of 56 analyzed carcinomas (15 follicular, 28 papillary, and 13 anaplastic) were clearly positive for BAG3 expression. CONCLUSIONS: BAG3 downmodulates the apoptotic response to TRAIL in human neoplastic thyroid cells. The protein is specifically expressed in thyroid carcinomas and not in normal thyroid tissue or goiter.

1-Methoxy-canthin-6-one induces c-Jun NH2-terminal kinase-dependent apoptosis and synergizes with tumor necrosis factor-related apoptosis-inducing ligand activity in human neoplastic cells of hematopoietic or endodermal origin.

We investigated the effects of 1-methoxy-canthin-6-one, isolated from the medicinal plant Ailanthus altissima Swingle, on apoptosis in human leukemia (Jurkat), thyroid carcinoma (ARO and NPA), and hepatocellular carcinoma (HuH7) cell lines. Cultures incubated with the compound showed >50% of sub-G1 (hypodiploid) elements in flow cytometry analysis; the apoptosis-inducing activity was evident at <10 micromol/L and half-maximal at about 40 micromol/L 1-methoxy-canthin-6-one. The appearance of hypodiploid elements was preceded by mitochondrial membrane depolarization, mitochondrial release of cytochrome c, and Smac/DIABLO and procaspase-3 cleavage. We subsequently investigated the effect of 1-methoxy-canthin-6-one in combination with human recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the four cell lines. Suboptimal concentrations (10 micromol/L 1-methoxy-canthin-6-one and 0.25 ng/mL TRAIL, respectively) of the two agents, unable to elicit apoptosis when used alone, induced mitochondrial depolarization, activation of caspase-3, and 45% to 85% of sub-G1 elements when added together to the cells. The synergism seemed to rely partly on the enhanced expression of TRAIL receptor 1 (TRAIL-R1; DR4), analyzed by immunofluorescence, by 1-methoxy-canthin-6-one. Cell incubation with 1-methoxy-canthin-6-one resulted in activating c-Jun NH2-terminal kinase (JNK), as revealed by Western blotting; induction of apoptosis and TRAIL-R1 up-regulation by 1-methoxy-canthin-6-one were >80% prevented by the addition of the JNK inhibitor (JNKI) SP600125JNKI, indicating that both effects were almost completely mediated by JNK activity. On the other hand, synergism with TRAIL was reduced by about 50%, suggesting that besides up-regulating TRAIL-R1, 1-methoxy-canthin-6-one could influence other factor(s) that participated in TRAIL-induced apoptosis. These findings indicate that 1-methoxy-canthin-6-one can represent a candidate for in vivo studies of monotherapies or combined antineoplastic therapies.

Proapoptotic effect of Uncaria tomentosa extracts.

Uncaria tomentosa ("Uña de gato") (Rubiaceae) is widely used in South America for treatment of gastritis, arthritis, cancer and inflammatory conditions. Recent literature reports cytostatic, antiproliferative, anti-inflammatory, mutagenic and anti-mutagenic properties of extracts of the plant. The present study investigates the possible proapoptotic mechanism via the activation of caspase3, in cytostatic effects of root bark extracts of Uncaria tomentosa on three different tumoral cell lines.

The anti-human leukocyte antigen-DR monoclonal antibody 1D09C3 activates the mitochondrial cell death pathway and exerts a potent antitumor activity in lymphoma-bearing nonobese diabetic/severe combined immunodeficient mice.

The fully human anti-HLA-DR antibody 1D09C3 has been shown to delay lymphoma cell growth in severe combined immunodeficient (SCID) mice. The present study was aimed at (a) investigating the mechanism(s) of 1D09C3-induced cell death and (b) further exploring the therapeutic efficacy of 1D09C3 in nonobese diabetic (NOD)/SCID mice. The chronic lymphocytic leukemia cell line JVM-2 and the mantle cell lymphoma cell line GRANTA-519 were used. Generation of reactive oxygen species (ROS) and mitochondrial membrane depolarization were measured by flow cytometry following cell incubation with dihydroethidium and TMRE, respectively. Western blot analysis was used to detect c-Jun-NH(2)-kinase (JNK) phosphorylation and apoptosis-inducing factor (AIF). NOD/SCID mice were used to investigate the activity of 1D09C3 in early- or advanced-stage tumor xenografts. In vitro, 1D09C3-induced cell death involves a cascade of events, including ROS increase, JNK activation, mitochondrial membrane depolarization, and AIF release from mitochondria. Inhibition of JNK activity significantly reduced 1D09C3-induced apoptosis, indicating that 1D09C3 activity involves activation of the kinase. In vivo, 1D09C3 induces long-term disease-free survival in a significant proportion of tumor-bearing mice treated at an early stage of disease. Treatment of mice bearing advanced-stage lymphoma results in a highly significant prolongation of survival. These data show that 1D09C3 (a) exerts a potent antitumor effect by activating ROS-dependent, JNK-driven cell death, (b) cures the great majority of mice treated at an early-stage of disease, and (c) significantly prolongs survival of mice with advanced-stage disease.

Dietary effects of copper and iron deficiency on rat intestine: a differential display proteome analysis.

Copper and iron are cofactors of many metallo-proteins that accomplish vital functions, such as oxygen and electron transport. Specific metabolic pathways have been selected through evolution, although still not fully elucidated, to confine the dangerous reactivity of their free ionic forms. Inadequate supply of both metals can severely affect basic physiological functions. A differential analysis of the rat intestinal proteome evidenced the following dietary copper- and iron-deficiencies, i.e., significant changes in the levels of proteins belonging to different functional classes (glucose and fatty acid metabolism, molecular chaperones, cytoskeleton plasticity, vitamin transporters). The presented results bring new perspectives to understand the role of copper and iron in the metabolic pathways and provide novel diagnostic tools to characterize the effects of subclinical deficiencies of both metals in unbalanced nutritional disorders.

Antiproliferative effects of tree-of-heaven (Ailanthus altissima Swingle).

Tree-of-heaven (Ailanthus altissima Swingle) was evaluated for its cytotoxic and antiproliferative activities by a bioassay-oriented study. Cytotoxicity observed in HeLa cells was time-dependent; the treatment with 10 microg/mL of the root chloroform extract reduced cell viability by 56% at 24 h and 29% at 48 h of exposure, whereas no effect was recorded in the controls. Significant effects were observed also for chromatographic fractions and the pure isolated alkaloid 1-methoxy-canthin-6-one. After 72 h of incubation cell viability was less than 10% for all treatments. A possible apoptotic effect was evaluated by monitoring the presence of hypodiploid elements in HeLa cells as well as in SAOS, U87MG and U-937 tumor cell lines. The cells incubated for different times with the active extract, fraction and pure alkaloid isolated from A. altissima showed a remarkable increase in the apoptosis.

Effect of unsaturated fat intake from Mediterranean diet on rat liver mRNA expression profile: selective modulation of genes involved in lipid metabolism.

BACKGROUND AND AIM: The lipid content of Mediterranean diet is mostly accounted for its disease preventive action. We investigated whether the short term nutritional effect of a fat quota mainly derived from olive and fish oil affects liver mRNA expression profile in rats. METHODS AND RESULTS: The study was carried out using DNA microarray techniques. The effect was evaluated at liver mRNA expression level to identify genes whose expression was regulated by dietary modifications. Two groups of six rats were alternatively supplied for two weeks with either a control or with an experimental diet. Both diets were semisynthetic and isocaloric, with identical major nutrients composition (protein 20%, carbohydrates 56% and lipids 22% of total energy) being different only in the quality of fats. The lipid quota of the control diet contained exclusively saturated animal fats, derived from butter, while in the experimental diet some unsaturated fats were present, being derived also from olive and fish oil (10% and 6% of total energy, respectively). Out of 26,334 genes analyzed, 11,292 were found expressed in the liver, 72 were induced and 180 were inhibited from the experimental diet. Out of these, 33 of the induced and 59 of the inhibited species have a well known function. CONCLUSIONS: The diet with olive and fish oil modulates several genes related to lipolysis or lipogenesis and newly identified responders from other metabolisms. Some of these genes are also reported to be similarly modulated by the action of fibrates, but without the complete gene activation typical of these PPARalpha ligands.

Identification of dietary copper- and iron-regulated genes in rat intestine.

Copper and iron act at different levels on gene expression. Due to their chemical reactivity, both metals could play a role in the regulation of the protein machinery involved in their metabolism, and/or of the metabolic function they are involved in. Experimental and clinical evidences raise also the hypothesis of the existence of genes commonly regulated by both metals. Purpose of this work was to find genes modulated by copper and iron in the rat intestine. A panel of 24 animals was randomly divided into three nutritional treatments including a control, a copper-deficient and an iron-deficient diet. The positive regulation of iron responsive element (IRE)-DMT1 gene was found, with different extent, in both experimental groups. A differential display reverse transcription (DDRT)-polymerase chain reaction (PCR) analysis carried out on the rat intestinal mRNAs demonstrated the differential expression of five cDNA fragments. Among these, the Cytochrome c oxidase (COX) subunit II mitochondrial gene resulted to be regulated by both metals, the Serum and Glucocorticoids-regulated Kinase (SGK) gene mainly by iron, and an Ebnerin-like 2 kb mRNA dramatically down-regulated by copper. Two residual clones showed low identity scores with sequences present in data bank. Finally, we observed that both iron and copper are able to modulate the expression of the three characterized genes in some tissues, other than intestine.

Regulation by heavy metals and temperature of the human BAG-3 gene, a modulator of Hsp70 activity.

In HeLa cells the expression of the BAG-3 gene, a member of the BAG family, is regulated by heavy metals and temperature, with kinetics of accumulation of mRNAs similar to Hsp70 and metallothioneins. Western blot assays performed with a polyclonal anti-BAG-3 antibody confirmed that higher levels of the protein were present in the cells following heat and metal exposure. By immunofluorescence techniques and cell fractionation assays we demonstrated that following stress BAG-3 protein concentrated in the rough endoplasmic reticulum and associated with the heavy membrane fraction. The role of BAG-3 protein during apoptosis and cellular stress is discussed.