Resveratrol differentially regulates NAMPT and SIRT1 in hepatocarcinoma cells and primary human hepatocytes

Resveratrol is reported to possess chemotherapeutic properties in several cancers. In this study, we wanted to investigate the molecular mechanisms of resveratrol-induced cell cycle arrest and apoptosis as well as the impact of resveratrol on NAMPT and SIRT1 protein function and asked whether there are differences in hepatocarcinoma cells (HepG2, Hep3B cells) and non-cancerous primary human hepatocytes. We found a lower basal NAMPT mRNA and protein expression in hepatocarcinoma cells compared to primary hepatocytes. In contrast, SIRT1 was significantly higher expressed in hepatocarcinoma cells than in primary hepatocytes. Resveratrol induced cell cycle arrest in the S- and G2/M- phase and apoptosis was mediated by activation of p53 and caspase-3 in HepG2 cells. In contrast to primary hepatocytes, resveratrol treated HepG2 cells showed a reduction of NAMPT enzymatic activity and increased p53 acetylation (K382). Resveratrol induced NAMPT release from HepG2 cells which was associated with increased NAMPT mRNA expression. This effect was absent in primary hepatocytes where resveratrol was shown to function as NAMPT and SIRT1 activator. SIRT1 inhibition by EX527 resembled resveratrol effects on HepG2 cells. Furthermore, a SIRT1 overexpression significantly decreased both p53 hyperacetylation and resveratrol-induced NAMPT release as well as S-phase arrest in HepG2 cells. We could show that NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and primary hepatocytes and that resveratrol did not act as a SIRT1 activator in hepatocarcinoma cells

Drowning and Nonfatal Drowning in Children and Adolescents: A Subsequent Retrospective Data Analysis

Fatal and nonfatal drowning are among the leading causes of death and lifelong severe neurological impairment among children and adolescents. This study aimed to complement research from Leipzig 1994–2008 to seek trends within risk factors, treatments, and outcomes throughout the last decade. We retrospectively investigated data of 47 inpatients aged 0–18 admitted to Leipzig University Department of Pediatrics who matched ICD-10 code T75.1 from 2008 to 2020 and compared them to a preceding study at the same institution. We also examined the prognostic value of parameters regarding the patients’ outcomes. There were three median incidents per annum. The median age was 2.75 years; 76% of incidents happened in males. An accumulation was seen during the summer months and weekends. Most drowning incidents occurred in private ponds or pools (48.9%). Thirty-nine children were discharged without resulting morbidity, four showed neurological impairment, and three died. Risk factors concerning age, sex, and incident characteristics were confirmed. Special supervision needs still apply to 1–3-year-old male children or children with pre-existing health conditions around private pools and ponds. Hospitalization duration shortened, and morbidity and lethality decreased since the previous study. There was structural improvement in primary care and medical documentation. Parameters suggesting good outcomes include a submersion time 3 points, spontaneous movement upon admission, remaining pupillary light response, the absence of cardiovascular arrest, body temperature ≥ 32 °C, pH > 7, blood glucose < 15 mmol/L, lactate < 14 mmol/L, base excess ≥ −15 mmol/L, and the absence of ARDS. Clear legislation can contribute to improved private home water safety. Further studies should include a broad in- and outpatient spectrum and standardized incident documentation presupposing Utstein-style reporting. Regular reinvestigation of consistent geographical regions facilitates process evaluations of drowning epidemiology and therapy evolution

Inhibition of NAMPT sensitizes MOLT4 leukemia cells for etoposide treatment through the SIRT2-p53 pathway

NAMPT (Nicotinamide phosphoribosyltransferase) catalyses the rate-limiting step in the NAD biosynthesis from nicotinamide and thereby regulates the activity of NAD-dependent enzymes. Cancer cells are highly dependent on NAD for energy and DNA repair processes and are assumed to be more susceptible to an inhibition of NAD synthesis than non-transformed cells. We aimed to investigate whether or not inhibition of NAMPT with its specific inhibitor FK866 can sensitize leukemia cells for chemotherapeutic agents. NAMPT protein abundance, enzymatic activity and NAD concentrations were significantly higher in Jurkat and Molt-4 leukemia cell lines compared to normal peripheral blood mononuclear cells. Combination of etoposide and FK866 caused increased cell death in leukemia cell lines compared to etoposide alone. Etoposide decreased protein abundance of NAD-dependent deacetylases SIRTUIN1. After combining etoposide and FK866 treatment SIRTUIN2 was further decreased and accumulation and acetylation of the downstream target p53 was further enhanced in MOLT4 cells. Concomitantly, protein abundance of p21 and cleaved BAX was increased. Targeting NAMPT could be a novel therapeutic strategy to enhance the efficacy of chemotherapeutic agents such as etoposide against leukemia

SIRT1 overexpression in HepG2 cells reversed resveratrol-induced SIRT1 inhibition, NAMPT release and S-phase arrest.

<p>A) SIRT1 was transiently overexpressed in HepG2 cells [2.0 µg plasmid/0.5x10⁶ cells] using the expression vector pECE_Flag-SIRT1 from addgene (plasmid 1791; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091045#pone.0091045-Brunet1" target="_blank">[35]</a>). Lysates of cells transfected with the empty vector pECE (mock-control) (1) or pECE Flag-SIRT1 vector (2) were used for Western Blot analysis. B) mock-transfected (mock-control) and Flag-SIRT1 transfected HepG2 cells were stimulated with resveratrol [50, 100 µM Resv.] for 24 h and Western Blot analysis of acetylated p53 (K382), p21 and GAPDH was performed. Densitometric anaylsis of acetylated p53 of three independent Western Blots is shown. Data were normalised to non-transfected HepG2 cells stimulated with resveratrol alone which was set 1. C) To analyse the effect of SIRT1 overexpression on resveratrol-induced NAMPT release, supernatant of mock-transfected and Flag-SIRT1 transfected HepG2 cells stimulated with or without resveratrol [100 µM] were used to measure eNAMPT level. One representative Western blot out of 3 independent experiments is shown. D) Cell viability of mock-transfected and Flag-SIRT1 transfected HepG2 cells treated with resveratrol [100 µM] (black bars) was measured using WST-1 assay (n = 3). Data were normalised to untreated mock-control which was set 1. E) mock-transfected (white bars) and Flag-SIRT1 transfected HepG2 cells (black bars) were stimulated with resveratrol [25, 50 µM] for 24 h. Percentage of cells in the S-phase were measured by PI staining and FACS analysis. All data are shown as mean± SEM (n = 4). The difference between two groups was evaluated using unpaired Student’s <i>t</i>-test (##p<0.01 mock-transfected cells compared to mock-transfected cells treated with resveratrol (white bars, mock-control), **p<0.01, ***p<0.001 Flag-SIRT1 transfected cells treated with resveratrol (black bars) compared to resveratrol-treated mock-transfected cells (white bars).</p

SIRT1 inhibition downregulates NAMPT activity and induces NAMPT release in HepG2 cells.

<p>HepG2 cells were treated with EX527+TSA [20 µM EX527+1 µM TSA] or serum-free medium (con) for 24 h. Measurement of A) NAMPT enzymatic activity (n = 3). Counts (cpm) were normalised to µg total protein in each sample (*p<0.05). B) NAD level were determined by HPLC (n = 5) and normalised to total protein amount in each sample. C) Supernatant of EX527 treated HepG2 cells was used for determination of eNAMPT level. One representative Western blot out of 3 independent experiments is shown.</p

Sequences of Primer and Probes used for <i>real-time</i> PCR (TaqMan).

<p><i>NAMPT (</i>nicotinamide phosphoribosyltransferase, also known as PBEF, visfatin); <i>p21</i>; housekeeping genes <i>beta-ACTIN</i>, <i>TBP</i> (TATA-box-binding protein) and <i>HPRT</i> (hypoxanthine phophoribosyltransferase).</p

Resveratrol differentially regulates p53 acetylation and SIRT1 protein level in HepG2 cells and primary human hepatocytes.

<p>Acetylation of p53 (K382) in A) HepG2 cells (n = 4) and B) primary human hepatocytes (n = 3) was evaluated by Western Blot. Densitometric analysis of at least three independent experiments is shown. Data are represented as mean± SEM and statistical analysis was performed using one-way ANOVA and the Bonferroni post hoc test (*p<0.05, n.s. not significant). As a downstream target of acetylated and activated p53, the expression of p21 was analysed by Western Blot. As positive control for SIRT1 inhibition, EX527+TSA was used. SIRT1 protein expression was analysed by Western Blot in C) HepG2 cells and D) primary hepatocytes and densitometric analysis was performed. GAPDH was used as loading control, respectively. One representative blot out of at least 3 independent experiments is shown.</p

NAMPT and SIRT1 expression in hepatocarcinoma cells and primary human hepatocytes.

<p>A) mRNA expression and B) protein expression of NAMPT and SIRT1 in primary human hepatocytes (n = 7), HepG2 cells (n = 8) and Hep3B cells (n = 3). Representative Western Blot is shown out of three independent experiments. Measurement of C) intracellular NAD levels (left panel, primary hepatocytes n = 4, HepG2 cells n = 6), basal NAMPT enzymatic activity (middle panel, primary hepatocytes n = 3, HepG2 cells n = 4) and extracellular NAMPT (eNAMPT) levels (right panel, primary hepatocytes n = 3, HepG2 cells n = 6) in primary human hepatocytes and HepG2 cells. Data are shown as mean± SEM. Difference between two groups was evaluated using unpaired Student’s <i>t</i>-test (*p<0.05, **p<0.01, ***p<0.001).</p

Resveratrol activates apoptotic mechanisms in hepatocarcinoma cells.

<p>Cells were treated with resveratrol or serum-free medium (con) for 24 h. Activation of p53 through phosphorylation at serine residue 15 and cleavage of caspase-3 in A) HepG2 cells, B) Hep3B cells and C) primary human hepatocytes were analysed by Western Blot. GAPDH was used as loading control. One representative blot out of at least 3 independent experiments is shown.</p