Cytarabine induces Atg expression, inhibits mTOR and modulates Akt/ERK signaling in REH cells.

<p>(A, B) REH cells were incubated for the indicated time periods with cytarabine (3.2 μM) and the levels of phosphorylated/total mTOR, p70S6K, AMPK, Akt and ERK were determined by immunoblotting. The representative blots from three independent experiments are shown (A), while the densitometry data are mean ± SD values (*p<0.05 compared to untreated cells) (B). (C, D) REH cells were incubated for 16h (C) or indicated time periods (D) with cytarabine (3.2 μM) in the absence or presence of leucine (2 mM) (C). Intracellular acidification in acridine orange-stained cells was determined by flow cytometry (C), while the amounts of Atg4, Atg5, Atg7, Atg12 and p62 mRNA were analyzed by RT-PCR (D). The data are mean ± SD values from three independent experiments (C) or mean ± SD values of triplicates from a representative of three experiments (D) (*p<0.05 compared to untreated cells).</p

Cytarabine induces apoptosis in REH cells.

<p>(A) REH cells were incubated in the presence of different concentrations of cytarabine and cell viability was determined by acid phosphatase assay after 24 h and 48 h. The data are mean ± SD values of triplicate measurements from a representative of three independent experiments (*p<0.05 compared to untreated cells). (B) REH cells were incubated for 24 h with cytarabine (3.2 μM) and phosphatidylserine exposure was determined by flow cytometry after annexin/PI staining. The dot plots from a representative of three independent experiments are presented.</p

Pharmacological inhibition of autophagy enhances cytarabine-induced apoptosis in leukemic cells.

<p>REH (A-F), HL-60 (G) and PBMC from CML patients (H) or healthy controls (I) were incubated for 24 h with cytarabine (3.2 μM) in the presence or absence of the autophagy inhibitors bafilomycin A1 (BAF; 10 nM) or chloroquine (CQ; 20 μM). Cell viability was determined by acid phosphatase test (A, G-I), while DNA fragmentation (B), phosphatidylserine exposure (C), caspase activation (D), mitochondrial depolarization (E) or superoxide production (F) were examined by flow cytometry using appropriate fluorochromes. The data are mean ± SD values of triplicates from a representative of three experiments (A, G-I) or mean ± SD values from three independent experiments (B-F) (*p<0.05 or ^#p<0.05 compared to untreated cells or cells treated with cytarabine alone, respectively).</p

Cytarabine inhibits mTOR and modulates AMPK/Akt/ERK signaling in HL-60 and primary leukemic cells.

<p>HL-60 (A), PBMC from CML patients (B) or healthy controls (C) were incubated for the indicated time periods with cytarabine (3.2 μM) and the levels of phosphorylated/total mTOR, p70S6K, AMPK, Akt and ERK were determined by immunoblotting. The representative blots from three independent experiments (A) or three different PBMC samples (B, C) are shown, while the densitometry data are mean ± SD values (*p<0.05 compared to untreated cells).</p

Cytarabine induces autophagy in REH cells.

<p>REH cells were incubated with cytarabine alone (3.2 μM) (A-C, E), or with cytarabine in the absence or presence of proteolysis inhibitors bafilomycin A1 (Baf; 10 nM) or chloroquine (Cq; 20 μM) (D). After 16 h, the presence of acridine orange-stained intracellular vesicles was demonstrated by flow cytometry, showing an increase in red/green (FL3/FL1) mean fluorescence ratio (A), or fluorescent microscopy (B). LC3 conversion, beclin-1 and p62 levels were assessed by immunoblotting at the indicated time points (C) or after 8 h (D). The blots from a representative of three independent experiments are presented, while the densitometry data are mean ± SD values (*p<0.05 or ^#p< 0.05 compared to untreated cells or cells treated with proteolysis inhibitors, respectively). (E) The presence of autophagic vesicles was analyzed after 24 h by electron microscopy and the representative micrographs from two experiments are shown.</p

RNA interference-mediated p62 knockdown inhibits autophagy and increases apoptosis in cytarabine-treated REH cells.

<p>(A) REH cells were transfected with control, LC3β or p62 siRNA, and p62 and LC3 expression were estimated by immunoblot. (B-H) REH cells transfected with control or p62 siRNA were incubated for 24 h with cytarabine (3.2 µM). Cell viability was analyzed by acid phosphatase assay (C), while the presence of acridine orange (AO)-stained intracellular vesicles (B), DNA fragmentation (D), phosphatidylserine exposure (E), caspase activation (F), mitochondrial depolarization (G) or superoxide production (H) were determined by flow cytometry using appropriate fluorochromes. The data are mean ± SD values of triplicates from a representative of three experiments (C) or mean ± SD values from three independent experiments (B, D-H) (*p < 0.05 or ^#p < 0.05 compared to untreated or cytarabine-treated control siRNA-transfected cells, respectively).</p

Large Graphene Quantum Dots Alleviate Immune-Mediated Liver Damage

We investigated the effect of large (40 nm) graphene quantum dots (GQDs) in concanavalin A (Con A; 12 mg/kg i.v.)-induced mouse hepatitis, a T cell-mediated liver injury resembling fulminant hepatitis in humans. Intravenously injected GQDs (50 mg/kg) accumulated in liver and reduced Con A-mediated liver damage, as demonstrated by histopathological analysis and a decrease in liver lipid peroxidation and serum levels of liver transaminases. The cleavage of apoptotic markers caspase-3/PARP and mRNA levels of proapoptotic mediators Puma, Noxa, Bax, Bak1, Bim, Apaf1, and p21, as well as LC3-I conversion to autophagosome-associated LC3-II and expression of autophagy-related (Atg) genes Atg4b, Atg7, Atg12, and beclin-1, were attenuated by GQDs, indicating a decrease in both apoptosis and autophagy in the liver tissue. This was associated with the reduced liver infiltration of immune cells, particularly the T cells producing proinflammatory cytokine IFN-γ, and a decrease in IFN-γ serum levels. In the spleen of GQD-exposed mice, mRNA expression of IFN-γ and its transcription factor T-bet was reduced, while that of the IL-33 ligand ST2 was increased. The hepatoprotective effect of GQDs was less pronounced in ST2-deficient mice, indicating that it might depend on ST2 upregulation. <i>In vitro</i>, GQDs inhibited splenocyte IFN-γ production, reduced the activation of extracellular signal-regulated kinase in macrophage and T cell lines, inhibited macrophage production of the free radical nitric oxide, and reduced its cytotoxicity toward hepatocyte cell line HepG2. Therefore, GQDs alleviate immune-mediated fulminant hepatitis by interfering with T cell and macrophage activation and possibly by exerting a direct hepatoprotective effect