Jaime M. Merino

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.

Identification of an Aspartic Residue in the P-loop of the Vanilloid Receptor That Modulates Pore Properties

Vanilloid receptor subunit 1 (VR1) is a nonselective cation channel that integrates multiple pain-producing stimuli. VR1 channels are blocked with high efficacy by the well established noncompetitive antagonist ruthenium red and exhibit high permeability to divalent cations. The molecular determinants that define these functional properties remain elusive. We have addressed this question and evaluated by site-specific neutralization the contribution on pore properties of acidic residues located in the putative VR1 pore region. Mutant receptors expressed inXenopus oocytes exhibited capsaicin-operated ionic currents akin to those of wild type channels. Incorporation of glutamine residues at Glu648 and Glu651 rendered minor effects on VR1 pore attributes, while Glu636 slightly modulated pore blockade. In contrast, replacement of Asp646by asparagine decreased 10-fold ruthenium red blockade efficacy and reduced 4-fold the relative permeability of the divalent cation Mg2+ with respect to Na+ without changing the selectivity of monovalent cations. At variance with wild type channels and E636Q, E648Q, and E651Q mutant receptors, ruthenium red blockade of D646N mutants was weakly sensitive to extracellular pH acidification. Collectively, our results suggest that Asp646 is a molecular determinant of VR1 pore properties and imply that this residue may form a ring of negative charges that structures a high affinity binding site for cationic molecules at the extracellular entryway.

The Dioxin Receptor Regulates the Constitutive Expression of the Vav3 Proto-Oncogene and Modulates Cell Shape and Adhesion

The dioxin receptor (AhR) modulates cell plasticity and migration, although the signaling involved remains unknown. Here, we report a mechanism that integrates AhR into these cytoskeleton-related functions. Immortalized and mouse embryonic fibroblasts lacking AhR (AhR-/-) had increased cell area due to spread cytoplasms that reverted to wild-type morphology upon AhR re-expression. The AhR-null phenotype included increased F-actin stress fibers, depolarized focal adhesions, and enhanced spreading and adhesion. The cytoskeleton alterations of AhR-/- cells were due to down-regulation of constitutive Vav3 expression, a guanosine diphosphate/guanosine triphosphate exchange factor for Rho/Rac GTPases and a novel transcriptional target of AhR. AhR was recruited to the vav3 promoter and maintained constitutive mRNA expression in a ligand-independent manner. Consistently, AhR-/- fibroblasts had reduced Rac1 activity and increased activation of the RhoA/Rho kinase (Rock) pathway. Pharmacological inhibition of Rac1 shifted AhR+/+ fibroblasts to the null phenotype, whereas Rock inhibition changed AhR-null cells to the AhR+/+ morphology. Knockdown of vav3 transcripts by small interfering RNA induced cytoskeleton defects and changes in adhesion and spreading mimicking those of AhR-null cells. Moreover, vav3-/- MEFs, as AhR-/- mouse embryonic fibroblasts, had increased cell area and enhanced stress fibers. By modulating Vav3-dependent signaling, AhR could regulate cell shape, adhesion, and migration under physiological conditions and, perhaps, in certain pathological states.

Arginine-rich peptides are blockers of VR-1 channels with analgesic activity

Vanilloid receptors (VRs) play a fundamental role in the transduction of peripheral tissue injury and/or inflammation responses. Molecules that antagonize VR channel activity may act as selective and potent analgesics. We report that synthetic arginine‐rich hexapeptides block heterologously expressed VR‐1 channels with submicromolar efficacy in a weak voltage‐dependent manner, consistent with a binding site located near/at the entryway of the aqueous pore. Dynorphins, natural arginine‐rich peptides, also blocked VR‐1 activity with micromolar affinity. Notably, synthetic and natural arginine‐rich peptides attenuated the ocular irritation produced by topical capsaicin application onto the eyes of experimental animals. Taken together, our results imply that arginine‐rich peptides are VR‐1 channel blockers with analgesic activity. These findings may expand the development of novel analgesics by targeting receptor sites distinct from the capsaicin binding site.

Small peptides patterned after the N‐terminus domain of SNAP25 inhibit SNARE complex assembly and regulated exocytosis

Synthetic peptides patterned after the C‐terminus of synaptosomal associated protein of 25 kDa (SNAP25) efficiently abrogate regulated exocytosis. In contrast, the use of SNAP25 N‐terminal‐derived peptides to modulate SNAP receptors (SNARE) complex assembly and neurosecretion has not been explored. Here, we show that the N‐terminus of SNAP25, specially the segment that encompasses 22Ala‐44Ile, is essential for the formation of the SNARE complex. Peptides patterned after this protein domain are potent inhibitors of SNARE complex formation. The inhibitory activity correlated with their propensity to adopt an α‐helical secondary structure. These peptides abrogated SNARE complex formation only when added previous to the onset of aggregate assembly. Analysis of the mechanism of action revealed that these peptides disrupted the binary complex formed by SNAP25 and syntaxin. The identified peptides inhibited Ca2+‐dependent exocytosis from detergent‐permeabilized excitable cells. Noteworthy, these amino acid sequences markedly protected intact hippocampal neurones against hypoglycaemia‐induced, glutamate‐mediated excitotoxicity with a potency that rivaled that displayed by botulinum neurotoxins. Our findings indicate that peptides patterned after the N‐terminus of SNAP25 are potent inhibitors of SNARE complex formation and neuronal exocytosis. Because of their activity in intact neurones, these cell permeable peptides may be hits for antispasmodic and analgesic drug development.

Aryl hydrocarbon receptor-dependent induction of apoptosis by 2,3,7,8-tetrachlorodibenzo-p-dioxin in cerebellar granule cells from mouse

J. Neurochem. (2011) 10.1111/j.1471‐4159.2011.07291.x

NMDA-induced neuroprotection in hippocampal neurons is mediated through the protein kinase A and CREB (cAMP-response element-binding protein) pathway.

N-Methyl-d-aspartate (NMDA) receptors play a critical role in the brain stimulating synaptic plasticity and mediating neurodegeneration; a neuroprotective role has also been described, but its molecular mechanisms in hippocampus are under study. Here, we report that in primary cultures of rat hippocampal neurons exposure to low micromolar NMDA concentrations are neuroprotective against excitotoxic insults, while high micromolar NMDA concentrations provoke neuronal death. Molecular analysis reveals that a toxic concentration of NMDA induced a transient phosphorylation of cAMP-response element-binding protein (pCREB) in 2 min that rapidly decreased below basal levels. In contrast, a nontoxic NMDA concentration gave up to longer (20 min) rise of pCREB, suggesting that neuroprotection could be associated to a relatively prolonged presence of pCREB in the neurons. In support of this tenet, rolipram, an inhibitor of phosphodiesterase IV that increases the levels of cAMP and pCREB, protected against NMDA-induced neuronal death. Similar results were obtained with dibutyrate-cAMP (a cAMP analogue with membrane permeability) that also abrogated NMDA excitotoxicity. Conversely, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H89), an inhibitor of protein kinase A (PKA), that prevents the formation of pCREB induced by nontoxic NMDA concentrations, reverted the neuroprotection achieved by preincubation of low micromolar NMDA concentrations. These results substantiate the notion that induction of pCREB via PKA plays an important role in NMDA-mediated neuroprotection.

Identification of SNARE complex modulators that inhibit exocytosis from an α-helix-constrained combinatorial library

Synthetic peptides patterned after the proteins involved in vesicle fusion [the so-called SNARE (soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins] are potent inhibitors of SNARE complex assembly and neuronal exocytosis. It is noteworthy that the identification of peptide sequences not related to the SNARE proteins has not been accomplished yet; this is due, in part, to the structural constraints and the specificity of the protein interactions that govern the formation of the SNARE complex. Here we have addressed this question and used a combinatorial approach to identify peptides that modulate the assembly of the SNARE core complex and inhibit neuronal exocytosis. An alpha-helix-constrained, mixture-based, 17-mer combinatorial peptide library composed of 137180 sequences was synthesized in a positional scanning format. Peptide mixtures were assayed for their ability to prevent the formation of the in vitro -reconstituted SDS-resistant SNARE core complex. Library deconvolution identified eight peptides that inhibited the assembly of the SNARE core complex. Notably, the most potent 17-mer peptide (acetyl-SAAEAFAKLYAEAFAKG-NH2) abolished both Ca2+-evoked catecholamine secretion from detergent-permeabilized chromaffin cells and L-glutamate release from intact hippocampal primary cultures. Collectively, these findings indicate that amino acid sequences that prevent SNARE complex formation are not restricted to those that mimic domains of SNARE proteins, thus expanding the diversity of molecules that target neuronal exocytosis. Because of the implication of neurosecretion in the aetiology of several human neurological disorders, these newly identified peptides may be considered hits for the development of novel anti-spasmodic drugs.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces apoptosis in neural growth factor (NGF)-differentiated pheochromocytoma PC12 cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent toxicant that alters normal brain development and produces cognitive disability and motor dysfunction. However, after decades of intense study, the molecular mechanisms of TCDD-induced neurotoxicity, the signaling pathways involved and its molecular targets in neurons still remain unknown. TCDD acts as an exogenous ligand of the aryl hydrocarbon receptor (AhR) that becomes a key signaling molecule in the regulation of the toxic and carcinogenic properties of TCDD. We have used NGF-differentiated pheochromocytoma (dPC12) cells to determine the type of cell death that takes place by TCDD toxicity. TCDD induced cell death in dPC12 cultures with an EC(50) of 218+/-24 nM, similar to that obtained in undifferentiated PC12 cells, 171+/-31 nM. Nuclear fragmentation was observed after TCDD incubation in parallel to an increase in caspase-3 activity. Staurosporine, which readily induced apoptosis in dPC12 cells, showed a similar increase in caspase-3 activity and the characteristic pattern of nuclear fragmentation. Flow cytometry measurements showed that dPC12 cells in the presence of TCDD were positive for annexin V labeling but negative for propidium iodide staining. In addition, TCDD increased the area of the peak corresponding to hypodiploid (apoptotic) DNA content. All together these results support the hypothesis that TCDD toxicity in dPC12 cells takes place mainly through an apoptotic process.

Thermal unfolding of monomeric Ca(II), Mg(II)-ATPase from sarcoplasmic reticulum of rabbit skeletal muscle.

The thermal unfolding of monomeric and delipidated Ca2+‐ATPase, solubilized in C12E8, can be appropriately described as a non‐two‐state irreversible denaturation, with only one endothermic peak. In the Ca2+ concentration range (0–0.5 mM) which stimulates the ATPase activity of solubilized monomeric ATPase, Ca2+ shifts the critical temperature midpoint of the denaturation process (T m) from 42 to 50°C without segregation of the endothermic peak into two separate components. Because 20 mM Mg2+ only shifts the T m from 42 to 44°C, we conclude that the effect of Ca2+ upon the T m is likely to be due to binding to the high affinity Ca2+ sites in the ATPase. The effect of Ca2+ upon the enthalpy of denaturation is biphasic, suggesting the presence of low affinity Ca2+ sites (K 0.5 in the millimolar range) in monomeric and solubilized ATPase.