Reticulon protein-1C is a key component of MAMs.

The endoplasmic reticulum (ER) is a key organelle fundamental for the maintenance of cellular homeostasis and the determination of cell fate under stress conditions. Reticulon-1C (RTN-1C) is a member of the reticulon family proteins localized primarily on the ER membrane and known to regulate ER structure and function. Several cellular processes depend on the structural and functional crosstalk between different organelles, particularly on the endoplasmic reticulum and mitochondria. These dynamic contacts, called mitochondria-associated ER membranes (MAMs), are essential for the maintenance of mitochondrial structure and participate in lipid and calcium exchanges between the two organelles. In this study we investigated the impact of RTN-1C modulation on mitochondrial dynamics. We demonstrate that RTN-1C controls mitochondrial structure and function affecting intracellular Ca2+ homeostasis and lipid exchange between ER and mitochondria. We propose that these events depend on RTN-1C involvement in the regulation of ER-mitochondria cross-talk and define a role for RTN-1C in maintaining the function of contacts between the two organelles.

SIRT1 silencing confers neuroprotection through IGF-1 pathway activation.

The following study demonstrated that, in in vitro differentiated neurons, SIRT1 silencing induced an increase of IGF-1 protein expression and secretion and of IGF-1R protein levels which, in turn, prolonged neuronal cell survival in presence of an apoptotic insult. On the contrary, SIRT1 overexpression increased cell death. In particular, IGF-1 and IGF-1R expression levels were negatively regulated by SIRT1. In SIRT1 silenced cells, the increase in IGF-1 and IGF-1R expression was associated to an increase in AKT and ERK1/2 phosphorylation. Moreover, neuronal differentiation was reduced in SIRT1 overexpressing cells and increased in SIRT1 silenced cells. We conclude that SIRT1 silenced neurons appear more committed to differentiation and more resistant to cell death through the activation of IGF-1 survival pathway.

Role of hypoxia and autophagy in MDA-MB-231 invasiveness.

Survival strategies adopted by tumor cells in response to a hypoxic stress include activation of hypoxia-inducible factor 1 (HIF-1) and autophagy. However, the importance and the function of each molecular response is not well defined. In the present study, we investigated invasiveness, migration, matrix metalloproteinases (MMPs) activity, and cell survival of MDA-MB-231 cells under normoxia, hypoxia, and hypoxia/reoxygenation (H/R). Moreover, to assess the importance of hypoxia and autophagy on the parameters studied, cells were either left untreated or treated with Chetomin (a selective inhibitor of HIF-1alpha) or trifluoperazine (TFP, an activator of autophagy). We found that hypoxia and H/R stimulated invasiveness and migration of MDA-MB-231 cells with an increased MMP-2 activity. Chetomin and TFP differently regulated the cellular behavior under the oxygenation conditions studied. In fact, Chetomin was most effective in inhibiting cell invasion, MMPs activity, and cell survival under hypoxia but not normoxia or H/R. By contrast, TFP inhibition of cell invasion, migration, and cell survival was independent from oxygenation conditions. TFP-induced autophagy was inhibited by light chain protein 3 (LC3) silencing or 3-methyladenine (3MA) treatment. In fact, LC3-silenced cells were able to invade in the presence of TFP without any GATE16 processing and p62 degradation. Immunofluorescence assay showed that LC3 silencing inhibited TFP-induced autophagosome formation. However, we also showed that both TPF treatment and LC3 silencing caused cytoskeleton impairments suggesting a possible interaction between LC3 and cytoskeleton components. In conclusion, our study shows that hypoxia and autophagy by acting on common (HIF-1alpha) or separate (MMPs, cytoskeleton) targets differently regulate cell invasion, MMPs activity, and survival.

Kaempferol induces apoptosis in two different cell lines via Akt inactivation, Bax and SIRT3 activation, and mitochondrial dysfunction.

Kaempferol (3,4',5,7-tetrahydroxyflavone) is a flavonoid with anti- and pro-oxidant activity present in various natural sources. Kaempferol has been shown to posses anticancer properties through the induction of the apoptotic program. Here we report that treatment of the chronic myelogenous leukemia cell line K562 and promyelocitic human leukemia U937 with 50 microM kaempferol resulted in an increase of the antioxidant enzymes Mn and Cu/Zn superoxide dismutase (SOD). Kaempferol treatment induced apoptosis by decreasing the expression of Bcl-2 and increasing the expressions of Bax. There were also induction of mitochondrial release of cytochrome c into cytosol and significant activation of caspase-3, and -9 with PARP cleavage. Kaempferol treatment increased the expression and the mitochondria localization of the NAD-dependent deacetylase SIRT3. K562 cells stably overexpressing SIRT3 were more sensitive to kaempferol, whereas SIRT3 silencing did not increase the resistance of K562 cells to kaempferol. Inhibition of PI3K and de-phosphorylation of Akt at Ser473 and Thr308 was also observed after treating both K562 and U937 cells with kaempferol. In conclusion our study shows that the oxidative stress induced by kaempferol in K562 and U937 cell lines causes the inactivation of Akt and the activation of the mitochondrial phase of the apoptotic program with an increase of Bax and SIRT3, decrease of Bcl-2, release of cytochrome c, caspase-3 activation, and cell death.

ERK-1 MAP kinase prevents TNF-induced apoptosis through bad phosphorylation and inhibition of Bax translocation in HeLa Cells.

Extracellular signal-regulated kinase (ERK) 1/2 signaling is involved in tumor cell survival through the regulation of Bcl-2 family members. To explore this further and to demonstrate the central role of the mitochondria in the ERK1/2 pathway we used the HeLa cellular model where apoptosis was induced by tumor necrosis factor (TNF) and cycloheximide (CHX). We show that HeLa cells overexpressing ERK-1 displayed resistance to TNF and CHX. HeLa cells overexpressing a kinase-deficient form of ERK-1 (K71R) were more sensitive to TNF and CHX. In the ERK-1 cells, Bad was phosphorylated during TNF + CHX treatment. In the HeLa wt cells and in the K71R clones TNF and CHX decreased Bad phosphorylation. ERK-1 cells treated with TNF and CHX did not release cytochrome c from the mitochondria. By contrast, HeLa wt and K71R clones released cytochrome c. Bax did not translocate to the mitochondria in ERK-1 cells treated with TNF + CHX. Conversely, HeLa wt and K71R clones accumulated Bax in the mitochondria. In the HeLa wt cells and in both ERK-1 transfectants Bid was cleaved and accumulated in the mitochondria. The caspase-8 inhibitor IETD-FMK and the mitochondrial membrane permeabilization inhibitor bongkrekic acid (BK), partially prevented cell death by TNF + CHX. Anisomycin, a c-Jun N-terminal kinases activator, increased TNF-killing. The ERK-1 cells were resistant to TNF and anisomycin, whereas K71R clones resulted more sensitive. Our study demonstrates that in HeLa cells the ERK-1 kinase prevents TNF + CHX apoptosis by regulating the intrinsic mitochondrial pathway through different mechanisms. Inhibition of the intrinsic pathway is sufficient to almost completely prevent cell death.

Transglutaminase 2 is involved in autophagosome maturation.

Autophagy is a highly conserved cellular process responsible for the degradation of long-lived proteins and organelles. Autophagy occurs at low levels under normal conditions, but it is enhanced in response to stress, e.g. nutrient deprivation, hypoxia, mitochondrial dysfunction and infection. "Tissue" transglutaminase (TG2) accumulates, both in vivo and in vitro, to high levels in cells under stressful conditions. Therefore, in this study, we investigated whether TG2 could also play a role in the autophagic process. To this end, we used TG2 knockout mice and cell lines in which the enzyme was either absent or overexpressed. The ablation of TG2 protein both in vivo and in vitro, resulted in an evident accumulation of microtubule-associated protein 1 light chain 3 cleaved isoform II (LC3 II) on pre-autophagic vesicles, suggesting a marked induction of autophagy. By contrast, the formation of the acidic vesicular organelles in the same cells was very limited, indicating an impairment of the final maturation of autophagolysosomes. In fact, the treatment of TG2 proficient cells with NH4Cl, to inhibit lysosomal activity, led to a marked accumulation of LC3 II and damaged mitochondria similar to what we observed in TG2-deficient cells. These data indicate a role for TG2-mediated post-translational modifications of proteins in the maturation of autophagosomes accompanied by the accumulation of many damaged mitochondria.