Unsupervised learning of aging principles from longitudinal data.

Age is the leading risk factor for prevalent diseases and death. However, the relation between age-related physiological changes and lifespan is poorly understood. We combined analytical and machine learning tools to describe the aging process in large sets of longitudinal measurements. Assuming that aging results from a dynamic instability of the organism state, we designed a deep artificial neural network, including auto-encoder and auto-regression (AR) components. The AR model tied the dynamics of physiological state with the stochastic evolution of a single variable, the "dynamic frailty indicator" (dFI). In a subset of blood tests from the Mouse Phenome Database, dFI increased exponentially and predicted the remaining lifespan. The observation of the limiting dFI was consistent with the late-life mortality deceleration. dFI changed along with hallmarks of aging, including frailty index, molecular markers of inflammation, senescent cell accumulation, and responded to life-shortening (high-fat diet) and life-extending (rapamycin) treatments.

Aberrant upregulation of the glycolytic enzyme PFKFB3 in CLN7 neuronal ceroid lipofuscinosis.

CLN7 neuronal ceroid lipofuscinosis is an inherited lysosomal storage neurodegenerative disease highly prevalent in children. CLN7/MFSD8 gene encodes a lysosomal membrane glycoprotein, but the biochemical processes affected by CLN7-loss of function are unexplored thus preventing development of potential treatments. Here, we found, in the Cln7∆ex2 mouse model of CLN7 disease, that failure in autophagy causes accumulation of structurally and bioenergetically impaired neuronal mitochondria. In vivo genetic approach reveals elevated mitochondrial reactive oxygen species (mROS) in Cln7∆ex2 neurons that mediates glycolytic enzyme PFKFB3 activation and contributes to CLN7 pathogenesis. Mechanistically, mROS sustains a signaling cascade leading to protein stabilization of PFKFB3, normally unstable in healthy neurons. Administration of the highly selective PFKFB3 inhibitor AZ67 in Cln7∆ex2 mouse brain in vivo and in CLN7 patients-derived cells rectifies key disease hallmarks. Thus, aberrant upregulation of the glycolytic enzyme PFKFB3 in neurons may contribute to CLN7 pathogenesis and targeting PFKFB3 could alleviate this and other lysosomal storage diseases.

Targeting PFKFB3 alleviates cerebral ischemia-reperfusion injury in mice.

The glycolytic rate in neurons is low in order to allow glucose to be metabolized through the pentose-phosphate pathway (PPP), which regenerates NADPH to preserve the glutathione redox status and survival. This is controlled by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), the pro-glycolytic enzyme that forms fructose-2,6-bisphosphate, a powerful allosteric activator of 6-phosphofructo-1-kinase. In neurons, PFKFB3 protein is physiologically inactive due to its proteasomal degradation. However, upon an excitotoxic stimuli, PFKFB3 becomes stabilized to activate glycolysis, thus hampering PPP mediated protection of redox status leading to neurodegeneration. Here, we show that selective inhibition of PFKFB3 activity by the small molecule AZ67 prevents the NADPH oxidation, redox stress and apoptotic cell death caused by the activation of glycolysis triggered upon excitotoxic and oxygen-glucose deprivation/reoxygenation models in mouse primary neurons. Furthermore, in vivo administration of AZ67 to mice significantly alleviated the motor discoordination and brain infarct injury in the middle carotid artery occlusion ischemia/reperfusion model. These results show that pharmacological inhibition of PFKFB3 is a suitable neuroprotective therapeutic strategy in excitotoxic-related disorders such as stroke.

The abietane diterpenoid parvifloron D from Plectranthus ecklonii is a potent apoptotic inducer in human leukemia cells.

BACKGROUND: Abietane diterpenes have attracted much attention because they display a wide range of biological activities, including antitumor activities. These compounds are the most diverse of the diterpenoids isolated from species of Plectranthus. Naturally occurring diterpene parvifloron D is the main phytochemical constituent of Plectranthus ecklonii. To examine the therapeutic potential of the plant, we evaluated whether parvifloron D displays cytotoxicity against human tumor cells. METHODS: The cytotoxicity was analyzed by colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptosis was evaluated by fluorescent microscopy, transmission electron microscopy, flow cytometric analysis of annexin V-FITC and propidium iodide-stained cells and DNA fragmentation. Protein expression and processing and release of mitochondrial proteins were analyzed by Western blot. Caspase activity was determined using colorimetric substrates. The membrane potential and intracellular reactive oxygen species were detected by flow cytometry. RESULTS: Parvifloron D displays strong cytotoxic properties against leukemia cells (HL-60, U-937, MOLT-3 and K-562) and in particular P-glycoprotein-overexpressing K-562/ADR cells, but has only weak cytotoxic effects on peripheral blood mononuclear cells (PBMCs). Overexpression of the protective mitochondrial proteins Bcl-2 and Bcl-xL did not confer resistance to parvifloron D-induced cytotoxicity. Growth inhibition of HL-60 cells that was triggered by parvifloron D was found to be caused by a rapid induction of apoptotic cell death. This apoptosis was prevented by the non-specific caspase inhibitor z-VAD-fmk, and by the selective caspase-9 inhibitor z-LEHD-fmk. Cell death induced by parvifloron D was found to be (i) associated with the dissipation of the mitochondrial membrane potential and the release of cytochrome c, (ii) amplified by inhibition of extracellular signal-regulated kinases (ERKs) 1/2 signaling and (iii) caused by a mechanism dependent on intracellular reactive oxygen species generation. CONCLUSION: Parvifloron D is a potent cytotoxic compound against several human tumor cells and also a fast and potent apoptotic inducer in leukemia cells.

Synthesis and effects on cell viability of flavonols and 3-methyl ether derivatives on human leukemia cells.

Flavonoids are polyphenolic compounds which display an array of biological activities and are considered potential antitumor agents. Here we evaluated the antiproliferative activity of selected synthetic flavonoids against human leukemia cell lines. We found that 4'-bromoflavonol (flavonol 3) was the most potent. This compound inhibited proliferation in a concentration-dependent manner, induced apoptosis and blocked cell cycle progression at the S phase. Cell death was found to be associated with the cleavage and activation of multiple caspases, the activation of the mitogen-activated protein kinase pathway and the up-regulation of two death receptors (death receptor 4 and death receptor 5) for tumor necrosis factor-related apoptosis-inducing ligand. Moreover, combined treatments using 4'-bromoflavonol and TRAIL led to an increased cytotoxicity compared to single treatments. These results provide a basis for further exploring the potential applications of this combination for the treatment of cancer.

Induction of G2/M phase arrest and apoptosis by the flavonoid tamarixetin on human leukemia cells.

Flavonoids are naturally occurring polyphenolic compounds which display a vast array of biological activities. In this study, we investigated the effects of tamarixetin on viability of human tumor cell lines and found that it was cytotoxic against leukemia cells and in particular P-glycoprotein-overexpressing K562/ADR cells. This compound inhibited proliferation in a concentration- and time-dependent manner, induced apoptosis and blocked cell cycle progression at G2 -M phase. This was associated with the accumulation of cyclin B1, Bub1 and p21(Cip1/Waf-1), changes in the phosphorylation status of cyclin B1, Cdk1, Cdc25C and MPM-2, and inhibition of tubulin polymerization. Moreover, cell death was found to be associated with cytochrome c release and cleavage of caspases and of poly(ADP-ribose) polymerase, and completely abrogated by the free-radical scavenger N-acetyl-L-cysteine. The sensitivity of leukemic cells to tamarixetin suggests that it should be considered for further preclinical and in vivo testing.

Antiproliferative activity of abietane diterpenoids against human tumor cells.

In the present study, the cytotoxicity of 30 diterpenoids with an abietane or a halimane skeleton was determined against five human tumor cell lines (HL-60, U937, Molt-3, SK-MEL-1, and MCF-7). Diterpenoids containing an abietane skeleton including taxodone (1) and taxodione (2), as well as the semisynthetic derivatives 12, 14, 15, 17, and 22, were the most cytotoxic compounds for human leukemia cells. Overexpression of the protective mitochondrial proteins Bcl-2 and Bcl-x(L) did not confer resistance to abietane diterpene-induced cytotoxicity. Studies performed on HL-60 cells indicated that growth inhibition triggered by compounds 1, 12, 14, and 15 was caused by induction of apoptosis. This was prevented by the nonspecific caspase inhibitor Z-VAD-FMK and, in the case of compounds 14 and 15, reduced by the selective caspase-8 inhibitor Z-IETD-FMK. Cell death induced by these abietane diterpenes was found to be associated with the release of mitochondrial proteins, including cytochrome c, Smac/DIABLO, and AIF (apoptosis-inducing factor), accompanied by dissipation of the mitochondrial membrane potential (ΔΨ), and modulated by inhibition of extracellular signal-regulated kinases signaling and the p38 mitogen-activated protein kinase pathway.

Astragalin heptaacetate-induced cell death in human leukemia cells is dependent on caspases and activates the MAPK pathway.

Flavonoids are naturally occurring polyphenolic compounds and are among the most promising anticancer agents. Here we demonstrate that the flavonoid derivative astragalin heptaacetate (AHA) induces cell death. This was prevented by the non-specific caspase inhibitors z-VAD-fmk and Q-VD-OPH, and reduced by the selective caspase-4 inhibitor z-LEVD-fmk. AHA-induced cell death was found to be: (i) associated with the release of cytochrome c, (ii) suppressed by the overexpression of Bcl-x(L), (iii) amplified by inhibition of extracellular signal-regulated kinases (ERKs) 1/2 and c-jun NH(2)-terminal kinases/stress activated protein kinases (JNK/SAPK) signaling, and (iv) completely abrogated by the free-radical scavenger N-acetyl-l-cysteine.