Alberto Álvarez-Barrientos

Resveratrol‐induced apoptosis in MCF‐7 human breast cancer cells involves a caspase‐independent mechanism with downregulation of Bcl‐2 and NF‐κB

Resveratrol (RES), a chemopreventive molecule, inhibits the proliferation of tumor cells of different etiologies. We previously showed that RES alters the cell cycle and induces apoptosis in MCF‐7 breast tumor cells by interfering with the estrogen receptor (ERaα)–dependent phosphoinositide 3‐kinase (PI3K) pathway. Here, we analyzed signaling downstream of PI3K, to understand the mechanisms of RES‐induced apoptosis. Apoptotic death by RES in MCF‐7 was mediated by Bcl‐2 downregulation since overexpression of this protein abolished apoptosis. Decreased Bcl‐2 levels were not related to cytochrome c release, activation of caspases 3/8 or poly(ADP‐ribose) polymerase proteolysis. However, RES decreased mitochondrial membrane potential and increased reactive oxygen species and nitric oxide production. NF‐κB, a regulator of Bcl‐2 expression, and calpain protease activity, a regulator of NF‐κB, were both inhibited by RES. The patterns for NF‐κB and calpain activities followed that of PI3K and were inhibited by LY294002. NF‐κB inhibition coincided with diminished MMP‐9 activity and cell migration. These data suggest that RES‐induced apoptosis in MCF‐7 could involve an oxidative, caspase‐independent mechanism, whereby inhibition of PI3K signaling converges to Bcl‐2 through NF‐κB and calpain protease activity. Therefore, Bcl‐2 and NF‐κB could be considered potential targets for the chemopreventive activity of RES in estrogen‐responsive tumor cells.

Hematopoietic mobilization in mice increases the presence of bone marrow-derived hepatocytes via in vivo cell fusion.

The mechanisms for in vivo production of bone marrow–derived hepatocytes (BMDHs) remain largely unclear. We investigated whether granulocyte colony–stimulating factor (G‐CSF)–mediated mobilization of hematopoietic cells increases the phenomenon. Recurrent liver injury in mice expressing green fluorescent protein (EGFP) in all hematopoietic‐derived cells was produced by 3 months of carbon tetrachloride (CCL4) injections. Histologically, there were necrotic foci with histiocyte‐rich infiltrates, but little oval cell proliferation. Subsequently, some animals were mobilized with G‐CSF for 1, 2, or 3 weeks. Animals were sacrificed 1 month after growth factor treatment. BMDH percentages were lower than previously reported, though G‐CSF mobilization significantly augmented BMDH production in injured livers. BMDHs originating from in vivo fusion were evaluated by transplanting female EGFP+ cells into male mice. Binucleated, EGFP+ hepatocytes with one Y chromosome, indicating fusion, were identified. In conclusion, (1) mobilization of hematopoietic cells increases BMDH production and (2) as with the FAH‐null model, the first model demonstrating hematopoietic/hepatocyte fusion, recurring CCl4‐induced injury has macrophage‐rich infiltrates, a blunted oval cell response, and a predominantly in vivo fusion process for circulating cell engraftment into the liver. These findings open the possibility of using hematopoietic growth factors to treat nonhematopoietic degenerative diseases. (HEPATOLOGY 2006;43:108–116.)

Discrepancies Between Nitroglycerin and NO-Releasing Drugs on Mitochondrial Oxygen Consumption, Vasoactivity, and the Release of NO

It has been generally acknowledged that the actions of glyceryl trinitrate (GTN) are a result of its bioconversion into NO. However, recent observations have thrown this idea into doubt, with many studies demonstrating that NO is present only when there are high concentrations of GTN. We have explored this discrepancy by developing a new approach that uses confocal microscopy to directly detect NO. Intracellular levels of NO in the rat aortic vascular wall have been compared with those present after incubation with 3 different NO donors (DETA-NO, 3-morpholinosydnonimine, and S-nitroso-N-acetylpenicillamine), endothelial activation with acetylcholine, or administration of GTN. We have also evaluated the relaxant effects of these treatments on isolated rings of aorta following activation of the enzyme soluble guanylyl cyclase and their inhibitory action on mitochondrial respiration, which is an index of the interaction of NO with the enzyme of the electron transport chain cytochrome C oxidase. In the case of the various NO donors and acetylcholine, we detected a concentration-dependent relationship in the intensity of vascular relaxation and degree of NO fluorescence and an increase in the Michaelis constant (Km) for O2. GTN did not produce similar effects, and although clinically relevant concentrations of this compound caused clear, concentration-related relaxations, there was neither any increase in NO-related fluorescence nor an augmented Km for O2. The nature of these differences suggests that these concentrations of GTN do not release free NO but probably a different species that, although it interacts with soluble guanylyl cyclase in vascular smooth muscle, does not inhibit O2 consumption by vascular mitochondria.

Phosphorylation of STIM1 at ERK1/2 target sites modulates store-operated calcium entry

Store-operated calcium entry (SOCE) is an important Ca2+ entry pathway that regulates many cell functions. Upon store depletion, STIM1, a transmembrane protein located in the endoplasmic reticulum (ER), aggregates and relocates close to the plasma membrane (PM) where it activates store-operated calcium channels (SOCs). Although STIM1 was early defined as a phosphoprotein, the contribution of the phosphorylation has been elusive. In the present work, STIM1 was found to be a target of extracellular-signal-regulated kinases 1 and 2 (ERK1/2) in vitro, and we have defined the ERK1/2-phosphorylated sites on the STIM1 sequence. Using HEK293 cells stably transfected for the expression of tagged STIM1, we found that alanine substitution mutants of ERK1/2 target sites reduced SOCE significantly, suggesting that phosphorylation of these residues are required to fully accomplish SOCE. Indeed, the ERK1/2 inhibitors PD184352 and PD0325901 decreased SOCE in transfected cells. Conversely, 12-O-tetradecanoylphorbol-13-acetate, which activates ERK1/2, enhanced SOCE in cells expressing wild-type tagged STIM1, but did not potentiate Ca2+ influx in cells expressing serine to alanine mutations in ERK1/2 target sites of STIM1. Alanine substitution mutations decreased Ca2+ influx without disturbing the aggregation of STIM1 upon store depletion and without affecting the relocalization in ER–PM punctae. However, our results suggest that STIM1 phosphorylation at ERK1/2 target sites can modulate SOCE by altering STIM1 binding to SOCs, because a significant decrease in FRET efficiency was observed between alanine substitution mutants of STIM1–GFP and ORAI1–CFP.

TLR4-Mediated Survival of Macrophages Is MyD88 Dependent and Requires TNF-α Autocrine Signalling

Modulation of macrophage survival is a critical factor in the resolution of inflammatory responses. Exposure to LPS protects innate immune cells against apoptosis, although the precise pathways responsible for prolongation of macrophage survival remain to be fully established. The goal of this study was to characterize the mechanism of TLR4-mediated survival of murine bone marrow-derived macrophages upon M-CSF withdrawal in more detail. Using a combination of knockout mice and pharmacological inhibitors allowed us to show that TLR4 and TLR2 stimulation promotes long-term survival of macrophages in a MyD88-, PI3K-, ERK-, and NF-κB-dependent manner. LPS-induced long-term, but not short-term, survival requires autocrine signaling via TNF-α and is facilitated by a general cytoprotective program, similar to that mediated by M-CSF. TLR4-mediated macrophage survival is accompanied by a remarkable up-regulation of specific cell surface markers, suggesting that LPS stimulation leads to the differentiation of macrophages toward a mixed macrophage/dendritic cell-like phenotype.

A mesenchymal-like phenotype and expression of CD44 predict lack of apoptotic response to sorafenib in liver tumor cells

The multikinase inhibitor sorafenib is the only effective drug in advanced cases of hepatocellular carcinoma (HCC). However, response differs among patients and effectiveness only implies a delay. We have recently described that sorafenib sensitizes HCC cells to apoptosis. In this work, we have explored the response to this drug of six different liver tumor cell lines to define a phenotypic signature that may predict lack of response in HCC patients. Results have indicated that liver tumor cells that show a mesenchymal‐like phenotype, resistance to the suppressor effects of transforming growth factor beta (TGF‐β) and high expression of the stem cell marker CD44 were refractory to sorafenib‐induced cell death in in vitro studies, which correlated with lack of response to sorafenib in nude mice xenograft models of human HCC. In contrast, epithelial‐like cells expressing the stem‐related proteins EpCAM or CD133 were sensitive to sorafenib‐induced apoptosis both in vitro and in vivo. A cross‐talk between the TGF‐β pathway and the acquisition of a mesenchymal‐like phenotype with up‐regulation of CD44 expression was found in the HCC cell lines. Targeted CD44 knock‐down in the mesenchymal‐like cells indicated that CD44 plays an active role in protecting HCC cells from sorafenib‐induced apoptosis. However, CD44 effect requires a TGF‐β‐induced mesenchymal background, since the only overexpression of CD44 in epithelial‐like HCC cells is not sufficient to impair sorafenib‐induced cell death. In conclusion, a mesenchymal profile and expression of CD44, linked to activation of the TGF‐β pathway, may predict lack of response to sorafenib in HCC patients.

CCAAT/enhancer binding protein β deficiency provides cerebral protection following excitotoxic injury

The CCAAT/enhancer-binding protein β (C/EBPβ, also known as CEBPB) was first identified as a regulator of differentiation and inflammatory processes in adipose tissue and liver. Although C/EBPβ was initially implicated in synaptic plasticity, its function in the brain remains largely unknown. We have previously shown that C/EBPβ regulates the expression of genes involved in inflammatory processes and brain injury. Here, we have demonstrated that the expression of C/EBPβ is notably increased in the hippocampus in a murine model of excitotoxicity. Mice lacking C/EBPβ showed a reduced inflammatory response after kainic acid injection, and exhibited a dramatic reduction in pyramidal cell loss in the CA1 and CA3 subfields of the hippocampus. These data reveal an essential function for C/EBPβ in the pathways leading to excitotoxicity-mediated damage and suggest that inhibitors of this transcription factor should be evaluated as possible neuroprotective therapeutic agents.

Lovastatin inhibits the extracellular-signal-regulated kinase pathway in immortalized rat brain neuroblasts

We have shown previously that lovastatin, a 3-hydroxy-3-methyl- glutaryl coenzyme A reductase inhibitor, induces apoptosis in spontaneously immortalized rat brain neuroblasts. In the present study, we analysed the intracellular signal transduction pathways by which lovastatin induces neuroblast apoptosis. We showed that lovastatin efficiently inhibited Ras activation, which was associated with a significant decrease in ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Lovastatin also decreased CREB phosphorylation and CREB-mediated gene expression. The effects of lovastatin on the Ras/ERK1/2/CREB pathway were time- and concentration-dependent and fully prevented by mevalonate. In addition, we showed that two MEK [MAPK (mitogen-activated protein kinase)/ERK kinase] inhibitors, PD98059 and PD184352, were poor inducers of apoptosis in serum-treated neuroblasts. However, these inhibitors significantly increased apoptosis induced by lovastatin treatment. Furthermore, we showed that pharmacological inhibition of both MEK and phosphoinositide 3-kinase activities was able to induce neuroblast apoptosis with similar efficacy as lovastatin. Our results suggest that lovastatin triggers neuroblast apoptosis by regulating several signalling pathways, including the Ras/ERK1/2 pathway. These findings might also contribute to elucidate the intracellular mechanisms involved in the central nervous system side effects associated with statin therapy.

Phosphorylation of STIM1 at ERK1/2 target sites regulates interaction with the microtubule plus-end binding protein EB1.

Summary STIM1 (stromal interaction molecule 1) is a key regulator of store-operated calcium entry (SOCE). Upon depletion of Ca2+ concentration within the endoplasmic reticulum (ER), STIM1 relocalizes at ER-plasma membrane junctions, activating store-operated calcium channels (SOCs). Although the molecular details for STIM1-SOC binding is known, the regulation of SOCE remains largely unknown. A detailed list of phosphorylated residues within the STIM1 sequence has been reported. However, the molecular pathways controlling this phosphorylation and its function are still under study. Using phosphospecific antibodies, we demonstrate that ERK1/2 mediates STIM1 phosphorylation at Ser575, Ser608 and Ser621 during Ca2+ store depletion, and that Ca2+ entry and store refilling restore phosphorylation to basal levels. This phosphorylation occurs in parallel to the dissociation from end-binding protein 1 (EB1), a regulator of growing microtubule ends. Although Ser to Ala mutation of residues 575, 608 and 621 showed a constitutive binding to EB1 even after Ca2+ store depletion, Ser to Glu mutation of these residues (to mimic the phosphorylation profile attained after store depletion) triggered full dissociation from EB1. Given that wild-type STIM1 and STIM1S575E/S608E/S621E activate SOCE similarly, a model is proposed to explain how ERK1/2-mediated phosphorylation of STIM1 regulates SOCE. This regulation is based on the phosphorylation of STIM1 to trigger dissociation from EB1 during Ca2+ store depletion, an event that is fully reversed by Ca2+ entry and store refilling.

The dioxin receptor has tumor suppressor activity in melanoma growth and metastasis

Melanoma is a highly metastatic and malignant skin cancer having poor rates of patient survival. Since the incidence of melanoma is steadily increasing in the population, finding prognostic and therapeutic targets are crucial tasks in cancer. The dioxin receptor (AhR) is required for xenobiotic-induced toxicity and carcinogenesis and for cell physiology and organ homeostasis. Yet, the mechanisms by which AhR affects tumor growth and dissemination are largely uncharacterized. We report here that AhR contributes to the tumor-stroma interaction, blocking melanoma growth and metastasis when expressed in the tumor cell but supporting melanoma when expressed in the stroma. B16F10 cells engineered to lack AhR (small hairpin RNA for AhR) exacerbated melanoma primary tumorigenesis and lung metastasis when injected in AhR+/+ recipient mice but not when injected in AhR- /- mice or when co-injected with AhR-/- fibroblasts in an AhR+/+ stroma. Contrary, B16F10 cells expressing a constitutively active AhR had reduced tumorigenicity and invasiveness in either AhR genetic background. The tumor suppressor role of AhR in melanoma cells correlated with reduced migration and invasion, with lower numbers of cancer stem-like cells and with altered levels of β1-integrin and caveolin1. Human melanoma cell lines with highest AHR expression also had lowest migration and invasion. Moreover, AHR expression was reduced in human melanomas with respect to nevi lesions. We conclude that AhR knockdown in melanoma cells requires stromal AhR for maximal tumor progression and metastasis. Thus, AhR can be a molecular marker in melanoma and its activity in both tumor and stromal compartments should be considered.