Rosa Carnuccio

Regulation of autophagy by cytoplasmic p53

Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that deletion, depletion or inhibition of p53 can induce autophagy in human, mouse and nematode cells subjected to knockout, knockdown or pharmacological inhibition of p53. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53−/− cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.

Glucocorticoids inhibit the induction of nitric oxide synthase in macrophages

The effect of glucocorticoids on the production of NO2- and NO by the macrophage cell line J774 was investigated. Stimulation of the cells with lipopolysaccharide (LPS) resulted in a time-dependent accumulation of NO2- in the medium, reaching a plateau after 48h. Concomitant incubation of the cells for 24h with dexamethasone (0.001-1.0 microM) or hydrocortisone (0.01-10.0 microM) caused a concentration-dependent inhibition of NO2- formation. The cytosol of J774 cells stimulated with LPS and IFN-gamma produced a time-dependent increase in the release of NO. This was blocked in a concentration-dependent manner by dexamethasone and hydrocortisone, but not progesterone, administered concomitantly with the immunological stimulus. None of these compounds had any effect on the release of NO once the enzyme had been induced. The inhibitory effect of hydrocortisone on NO formation was blocked by cortexolone. These data suggest that part of the anti-inflammatory and immunosuppressive actions of glucocorticoids is due to their inhibition of the induction of the NO synthase.

Control of autophagy by oncogenes and tumor suppressor genes

Multiple oncogenes (in particular phosphatidylinositol 3-kinase, PI3K; activated Akt1; antiapoptotic proteins from the Bcl-2 family) inhibit autophagy. Similarly, several tumor suppressor proteins (such as BH3-only proteins; death-associated protein kinase-1, DAPK1; the phosphatase that antagonizes PI3K, PTEN; tuberous sclerosic complex 1 and 2, TSC1 and TSC2; as well as LKB1/STK11) induce autophagy, meaning that their loss reduces autophagy. Beclin-1, which is required for autophagy induction acts as a haploinsufficient tumor suppressor protein, and other essential autophagy mediators (such as Atg4c, UVRAG and Bif-1) are bona fide oncosuppressors. One of the central tumor suppressor proteins, p53 exerts an ambiguous function in the regulation of autophagy. Within the nucleus, p53 can act as an autophagy-inducing transcription factor. Within the cytoplasm, p53 exerts a tonic autophagy-inhibitory function, and its degradation is actually required for the induction of autophagy. The role of autophagy in oncogenesis and anticancer therapy is contradictory. Chronic suppression of autophagy may stimulate oncogenesis. However, once a tumor is formed, autophagy inhibition may be a therapeutic goal for radiosensitization and chemosensitization. Altogether, the current state-of-the art suggests a complex relationship between cancer and deregulated autophagy that must be disentangled by further in-depth investigation.

Involvement of NF‐κB in the regulation of cyclooxygenase‐2 protein expression in LPS‐stimulated J774 macrophages

We investigated the involvement of NF‐κB in the regulation of COX‐2 protein expression and prostaglandin production in LPS‐stimulated J774 macrophages. Incubation of J774 cells with LPS (1 μg/ml) for 24 h caused an increase of COX‐2 protein expression and accumulation of both PGE2 and 6‐keto‐PGF1α in the cell culture medium. Ammonium pyrrolidinedithiocarbamate (APDC, 0.1, 1, 10 μM) and N‐α‐p‐tosyl‐l‐lysine chloromethylketone (TLCK, 1, 10, 100 μM), two inhibitors of NF‐κB activation, suppressed in a concentration‐dependent manner both LPS‐induced COX‐2 protein expression and prostanoid generation. Moreover, APDC and TLCK both inhibited the LPS‐induced increase of NF‐κB DNA binding activity and prevented IκB‐α degradation. Our results show for the first time that NF‐κB is involved in COX‐2 protein expression in LPS‐stimulated J774 macrophages and suggest that inhibitors of NF‐κB activation may represent a useful tool for the pharmacological control of inflammation.

Glucocorticoids induce the formation and release of anti-inflammatory and anti-phospholipase proteins into the peritoneal cavity of the rat.

1 Dexamethasone and hydrocortisone induce the release of anti‐phospholipase proteins into the peritoneal cavities of rats. 2 Adrenocorticotropic hormone (ACTH) also releases these proteins in normal but not in adrenalectomized rats. 3 Peritoneal lavage proteins were separated by ion‐exchange and size exclusion chromatography. The anti‐phospholipase activity occurred in four separate fractions with the major component having an apparent mol. wt. of 40 k. 4 Column fractions containing these anti‐phospholipase proteins had anti‐inflammatory effects in the rat carrageenin pleurisy model whereas other fractions were inactive. 5 The proteins appear to be identical to macrocortin and lipomodulin, the ‘second messengers’ of glucocorticoid hormone action on the arachidonate system.

Stimulation of autophagy by the p53 target gene Sestrin2.

The oncosuppressor protein p53 regulates autophagy in a dual fashion. The pool of cytoplasmic p53 protein represses autophagy in a transcription-independent fashion, while the pool of nuclear p53 stimulates autophagy through the transactivation of specific genes. Here we report the discovery that Sestrin2, a novel p53 target gene, is involved in the induction of autophagy. Depletion of Sestrin2 by RNA interference reduced the level of autophagy in a panel of p53-sufficient human cancer cell lines responding to distinct autophagy inducers. In quantitative terms, Sestrin2 depletion was as efficient in preventing autophagy induction as was the depletion of Dram, another p53 target gene. Knockout of either Sestrin2 or Dram reduced autophagy elicited by nutrient depletion, rapamycin, lithium or thapsigargin. Moreover, autophagy induction by nutrient depletion or pharmacological stimuli led to an increase in Sestrin2 expression levels in p53-proficient cells. In strict contrast, the depletion of Sestrin2 or Dram failed to affect autophagy in p53-deficient cells and did not modulate the inhibition of baseline autophagy by a cytoplasmic p53 mutant that was reintroduced into p53-deficient cells. We conclude that Sestrin2 acts as a positive regulator of autophagy in p53-proficient cells.

Cannabidiol inhibits inducible nitric oxide synthase protein expression and nitric oxide production in β-amyloid stimulated PC12 neurons through p38 MAP kinase and NF-κB involvement

In view of the pro-inflammatory scenario observed in Alzheimers disease, in the recent years anti-inflammatory drugs have been proposed as potential therapeutic agents. We have previously shown that cannabidiol, the main non-psychotropic component from Cannabis sativa, possess a variegate combination of anti-oxidant and anti-apoptotic effects that protect PC12 cells from Abeta toxicity. In parallel, cannabidiol has been described to have anti-inflammatory properties in acute models of inflammation but the possible inhibitory effect of cannabidiol on iNOS protein expression and nitrite production in the nitrosative stress induced by Abeta in neuronal cell-line is un-investigated. Stimulation of differentiated PC12 cells with Abeta (1-42) (1 microg/mL) for 36 h caused a significant increase of nitrite production, compared to un-stimulated cells, that was inhibited in a concentration-dependent manner by both the non-selective iNOS inhibitor, L-NAME (0.3-30 microM), and, at higher extent, by the selective iNOS inhibitor SMT (0.3-30 microM). CBD (10(-6) to 10(-4) M) inhibited both nitrite production and iNOS protein expression induced by Abeta (1-42). Cannabidiol effect was mediated through the inhibition of phosphorylated form of p38 MAP kinase and transcription factor nuclear factor-kappaB activation in a concentration-dependent manner. The here reported data increases our knowledge about the possible neuroprotective mechanism of cannabidiol, highlighting the importance of this compound to inhibit beta-amyloid induced neurodegeneration, in view of its low toxicity in humans.

The marijuana component cannabidiol inhibits β-amyloid-induced tau protein hyperphosphorylation through Wnt/β-catenin pathway rescue in PC12 cells

Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder. A massive accumulation of β-amyloid (Aβ) peptide aggregates has been proposed as pivotal event in AD. Aβ-induced toxicity is accompanied by a variegated combination of events including oxidative stress. The Wnt pathway has multiple actions in the cascade of events triggered by Aβ, and drugs that rescue Wnt activity may be considered as novel therapeutics for AD treatment. Cannabidiol, a non-psychoactive marijuana component, has been recently proposed as an antioxidant neuroprotective agent in neurodegenerative diseases. Moreover, it has been shown to rescue PC12 cells from toxicity induced by Aβ peptide. However, the molecular mechanism of cannabidiol-induced neuroprotective effect is still unknown. Here, we report that cannabidiol inhibits hyperphosphorylation of tau protein in Aβ-stimulated PC12 neuronal cells, which is one of the most representative hallmarks in AD. The effect of cannabidiol is mediated through the Wnt/β-catenin pathway rescue in Aβ-stimulated PC12 cells. These results provide new molecular insight regarding the neuroprotective effect of cannabidiol and suggest its possible role in the pharmacological management of AD, especially in view of its low toxicity in humans.

Prostaglandins prevent inducible nitric oxide synthase protein expression by inhibiting nuclear factor-κB activation in J774 macrophages

We investigated the effect of PGE2 and iloprost (a prostacyclin analogue) on inducible nitric oxide synthase (iNOS) protein expression and nuclear factor‐κB (NF‐κB) activation in lipopolysaccharide (LPS)‐stimulated J774 macrophages. Incubation of J774 cells with LPS (10 μg/ml) caused an increase of iNOS protein expression which was prevented in a concentration‐dependent fashion by PGE2 (0.1, 1, 10 μM) and iloprost (0.01, 0.1, 1 μM). Electrophoretic mobility shift assay indicated that both prostanoids blocked the activation of NF‐κB, a transcription factor necessary for NO synthase induction. PGE2 and iloprost also blocked disappearance of IκB‐α from cytosolic fraction and nuclear translocation of NF‐κB subunits p50 and p65. These results show for the first time that PGE2 and iloprost down‐regulate iNOS protein expression by inhibiting NF‐κB activation and suggest a negative feed‐back mechanism that may be important for limiting excessive or prolonged NO production in pathological events.

Hydroxytyrosol, a phenolic compound from virgin olive oil, prevents macrophage activation

We investigated the effect of hydroxytyrosol (HT), a phenolic compound from virgin olive oil, on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in J774 murine macrophages stimulated with lipopolysaccharide (LPS). Incubation of cells with LPS caused an increase in iNOS and COX-2 mRNA and protein level as well as ROS generation, which was prevented by HT. In addition, HT blocked the activation of nuclear factor-κB (NF-κB), signal transducer and activator of transcription-1α (STAT-1α) and interferon regulatory factor-1 (IRF-1). These results, showing that HT down-regulates iNOS and COX-2 gene expression by preventing NF-κB, STAT-1α and IRF-1 activation mediated through LPS-induced ROS generation, suggest that it may represent a non-toxic agent for the control of pro-inflammatory genes.