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Previously, we demonstrated that a plant steroid, diosgenin, altered cell cycle distribution and induced apoptosis in the human osteosarcoma 1547 cell line. The objective of this study was to investigate if the antiproliferative effect of diosgenin was similar for different human cancer cell lines such as laryngocarcinoma HEp-2 and melanoma M4Beu cells. Moreover, this work essentially focused on the mitochondrial pathway. We found that diosgenin had an important and similar antiproliferative effect on different types of cancer cells. In addition, our new results show that diosgenininduced apoptosis is caspase-3 dependent with a fall of mitochondrial membrane potential, nuclear localization of AIF and poly (ADP-ribose) polymerase cleavage. Diosgenin treatment also induces p53 activation and cell cycle arrest in the different cell lines studied.

BackgroundAutophagy is a major pathway of protein and organelle degradation in the lysosome. Autophagy exists at basal constitutive level and can be induced as a defense mechanism under stress conditions. Molecular relationships between autophagy and inflammation at the periphery were recently evidenced, highlighting a role of autophagy in the regulation of inflammation. Impairment of autophagy (with accumulation of autophagic vacuoles) and substantial inflammation are found in neurodegenerative diseases such as Alzheimer’s Disease (AD). However, the links between autophagy and inflammation in AD remain to be determined.MethodsHere, we examined the inflammatory reaction and autophagy in murine tri-cultures of neurons, astrocytes, and microglia. Tri-cultures were exposed to various inflammatory stresses (lipopolysaccharide (LPS), amyloid peptide (Aβ42) with or without cytokines) for 48 hours. Furthermore, the relationships between inflammation and autophagy were also analyzed in astrocyte- and microglia-enriched cultures. Data for multiple variable comparisons were analyzed by a one-way ANOVA followed by a Newman-keuls’ test.ResultsAβ42 induced a low inflammation without accumulation of acidic vesicles contrary to moderate or severe inflammation induced by LPS or the cytokine cocktail (IL-1β, TNF-α, and IL-6) or IL-1β alone which led to co-localization of p62 and LC3, two markers of autophagy, with acidic vesicles stained with Lyso-ID Red dye. Moreover, the study reveals a major role of IL-1β in the induction of autophagy in tri-cultures in the presence or absence of Aβ42. However, the vulnerability of the autophagic process in purified microglia to IL-1β was prevented by Aβ42.ConclusionThese findings show a close relationship between inflammation and autophagy, in particular a major role of IL-1β in the induction of the microglial autophagy which could be the case in AD. New therapeutic strategies could target inflammasome and autophagy in microglia to maintain its role in the amyloid immunosurveillance.

The roles of glycogen synthase kinase-3beta (GSK-3beta) and tau phosphorylation were examined in seven-day-old rats injected with the NMDA receptor antagonist (MK801) that is known to induce neuronal apoptosis. Immunoblot and immunohistochemical analysis of brain samples demonstrated a site-specific increase in tau phosphorylation associated with the relocalization of the protein to the nuclear/perinuclear region of apoptotic neurons. In addition, a tau 32-kDa fragment was detected, suggesting that tau was a target of intracellular proteolysis in MK801-treated brains. The proteolytically modified form of tau has reduced ability to bind to microtubules. GSK-3beta kinase assay and immunoblottings of active (tyrosine-216) and inactive (serine-9) forms of GSK-3beta revealed a rapid and transient increase in the kinase activity. Lithium chloride, a GSK-3beta inhibitor, prevented tau phosphorylation suggesting that tau phosphorylation is mediated by the activation of GSK-3beta. Confocal microscopy using double labelling of tau and GSK-3beta revealed that the activation of GSK-3beta in neurons was associated with early (2 h) nuclear translocation of tyrosine-216 GSK-3beta. The execution phase of neuronal apoptosis was accompanied by a selective phosphorylation of serine-9 and dephosphorylation of tyrosine-216 GSK-3beta. These findings demonstrate that in vivo, GSK-3beta kinase activation and nuclear translocation are early stress signals of neuronal apoptosis.

BackgroundIn recent years, studies have sought to understand the mechanisms involved in the alteration of autophagic flux in Alzheimer's disease (AD). Alongside the recent description of the impairment of lysosomal acidification, we wanted to study the relationships between inflammation and autophagy, two physiological components deregulated in AD. Therefore, a longitudinal study was performed in APPswePS1dE9 transgenic mice at three, six and twelve months of age.MethodsAutophagic markers (Beclin-1, p62 and LC3) and the activation of mammalian Target of Rapamycin (mTOR) signaling pathway were quantified by western blot. Cytokine levels (IL-1β, TNF-α and IL-6) were measured by ELISA. Transmission electron microscopy was performed to detect autophagic vacuoles. Mann-Whitney tests were used to compare wild-type (WT) versus APPswePS1dE9 mice. Longitudinal changes in parameters were analyzed with a Kruskal-Wallis test followed by a post-hoc Dunn’s test. Correlation between two parameters was assessed using a Spearman test.ResultsCompared to 12-month old WT mice, 12-month old APPswePS1dE9 mice had higher levels of IL-1β and TNF-α, a greater inhibition of the mTOR signaling pathway and lower levels of Beclin-1 expression both in cortex and hippocampus. Regarding the relationship of the various parameters in 12-month old APPswePS1dE9 mice, Beclin-1 rates were positively correlated with IL-1β and TNF-α levels. And, on the contrary, TNF-α levels were inversely correlated with the levels of mTOR activation. Altogether, these results suggest that inflammation could induce autophagy in APPswePS1dE9 mice. However, these transgenic mice displayed a large accumulation of autophagic vesicles within dystrophic neurons in cortex and hippocampus, indicating a terminal failure in the autophagic process.ConclusionsThis first demonstration of relationships between inflammation and autophagy in in vivo models of AD should be taken into account in new therapeutic strategies to prevent inflammation and/or stimulate autophagy in advanced neurodegenerative process such as AD.