Involvement of p53 in MIF-induced HIF-1 activation.

<p>(A and B) MDA-MB-231 cells and Saos-2 cells were exposed to 20% or 1% O₂ conditions with or without rhMIF treatment for 4 h and then harvested for immunoblot assay for p53 (A) and HIF-1α and HIF-1β protein (B). (C and D) MCF-7 cells and Saos-2 cells were transfected with p2.1 reporter and pRL-SV40 (C). MCF-7 cells transfected with pSUPER (EV) or pSUPER-p53(p53) were transfected with p2.1 reporter and pRL-SV40 (D). Cells were exposed to 20% and 1% O₂ conditions with or without rhMIF treatment and then harvested for luciferase assay. Fold induction of relative luciferase activity was calculated. A normalized mean value±S.D. of three independent transfections is shown as fold induction. *; <i>p</i><0.05 compared to respective control (ANOVA). (E) MCF-7 and MDA-MB-231 cells transfected with pFLAG-MIF-wt plasmid were exposed to 1% O₂ and then cells were harvested. 500 µg of lysates were incubated with anti-FLAG affinity agarose beads and captured protein was eluted and analyzed by Western blot with anti-p53 antibody. 50 µg of lysate were analyzed by Western blot with anti-p53 antibody.</p

Additional file 3: Figure S3. of The antioxidant N-acetyl cysteine suppresses lidocaine-induced intracellular reactive oxygen species production and cell death in neuronal SH-SY5Y cells

Results of HeLa cell-derivatives EB8 and HeEB1. (A) Oxygen consumption rate of HeLa cells, EB8 and HeEB1cells were demonstrated. (B) Graphic depiction of reactive oxygen species (ROS) production in HeLa cells and EB8 cells exposed to the indicated concentrations of lidocaine (0, 4, or 10 mM) for 6 h (n = 3). Data depict the ratio of ROS production in treated cells compared to that in the untreated control group (HeLa cells). (C) Activities of Caspase3/7 of HeLa cells and HeEB1 cells were demonstrated. (D) Levels of cell death were measured using an Annexin V-FITC Apoptosis Detection Kit evaluated by FACS were demonstrated. (E) Graphic depiction of the levels of cell death among treated and untreated cell populations. Cell death was evaluated by measuring the levels of lactate dehydrogenase (LDH) within culture supernatants (n = 3) in the presence or absence of 10 mM N-acetyl cysteine (NAC), 250 μM Trolox and 10 μM GGA. Control is LDH activity treated by lysis buffer. (F) Graphic depiction of reactive oxygen species (ROS) production in SH-SY5Y cells exposed to 4 mM) for 6 h (n = 3) in the presence or absence of 10 mM N-acetyl cysteine (NAC), 250 μM Trolox and 10 μM GGA. Data depict the ratio of ROS production in treated cells compared to that in the untreated control group. Data presented in A–E expressed as means ± standard deviations (SD). #p < 0.05 compared with the control cell population at the same time period. (PDF 587 kb

Effect of MIF on HIF-1-dependent gene expressions.

<p>(A) MCF-7 cells were transfected with the indicated plasmids and exposed to 20% or 1% O₂ for 12 h. Then total RNA was isolated. Expression of VEGF, GLUT1, HIF-1α, and 18S rRNA was analyzed by RT-PCR using specific primer pairs. (B, C, D, and E) MCF-7 cells were transfected with pRL-SV40 encoding <i>Renilla</i> luciferase, FLAG-tagged MIF expression vector (B, C, and E), and one of the following plasmids encoding firefly luciferase: HRE reporter p2.1 (<i>B, C, and E</i>) or <i>VEGF</i> promoter reporter pVEGF-KpnI-Luc (<i>D</i>). (F) Constructs encoding the GAL4 DNA-binding domain (amino acids 1–147) fused to the indicated amino acids of HIF-1α were analyzed for their ability to transactivate reporter gene GAL4E1bLuc containing five GAL4-binding sites. MCF-7 cells were co-transfected with pRL-SV40 (50 ng), GAL4E1bLuc (100 ng), GAL4-HIF-1α fusion protein expression plasmids (100 ng), and FLAG-tagged MIF expression vector or empty vector (EV) (200 ng). Cells were treated with 100 µM of DFX or the indicated dose of rhMIF (F) and exposed to 20% or 1% O₂ conditions for 18 h and then harvested. Results shown represent mean±S.D. of three independent transfections. *; <i>p</i><0.05 compared to respective controls without MIF treatment (ANOVA).</p

Involvement of MIF in HIF-1 regulation in MCF-7 cells.

<p>HIF-1α protein stabilization and transactivation activity induced by MIF is dependent on p53- and MAPK-dependent signaling pathway.</p

Effect of MIF on HIF-1 protein expression in MCF-7 cells.

<p>(A and B) MCF-7 cells were transfected with 2 µg of p3xFLAG-CMV-10 plasmid (EV) or pFLAG-MIF plasmids (A and B lane 4; 2 µg, B lane 3; 0.5 µg). (C) MCF-7 cells were transfected by the plasmids for expression of FLAG tag (EV) and FLAG-tagged MIF and selected by puromycin resistance. Two clones of MCF-7 cells stably expressing FLAG tag (EV) and FLAG-tagged MIF (MIF#1 and MIF#2 cells) were established. (D) MCF-7 cells were treated with the indicated doses of bacterially produced recombinant human (rh)MIF. (E) MCF-7 cells were treated with rhMIF. Cells were exposed to 20% (A, B, C, and D), 5% (C), or 1% (A, B, C, and D) O₂ conditions or treated with 100 µM DFX (E) for 4 h. Cell were harvested for immunoblot assay for MIF (A and C), HIF-1α and HIF-1β protein (A, B, C, D, and E). Representative immunoblots are shown. Intensity of respective bands were analyzed densitometrically and fold induction to respective controls (lane1) are plotted accordingly as mean±S.D. (n = 3) *<i>p</i><0.05 compared to 20% O₂ conditions without MIF (lane 1) (ANOVA).</p

Effect of MIF-gene silencing on HIF-1 activity.

<p>(A, B, and D) MCF-7 cells were transfected with siRNA against MIF, TRX, CD74, or green fluorescence protein (GFP) as a negative control. Cells were exposed to 20% or 1% O₂ conditions and were harvested for immunoblot assay for MIF or TRX (left panels) or HIF-1α and HIF-1β protein expression (right panels). Representative immunoblots are shown. Intensity of respective bands were analyzed densitometrically and fold induction to lane 1 are plotted accordingly as mean±S.D. (n = 3) (A, B, and C). *<i>p</i><0.05 compared to 20% O₂ conditions without treatment (ANOVA). (C) MCF-7 cells were pre-treated with 10 µM α-tocopherol or 10 mM NAC and then exposed to 20% or 1% O₂ conditions for 4 h. Cells were harvested for immunoblot assay for HIF-1α and HIF-1β protein. (D) siRNAs for GFP or CD74 were introduced into MCF-7 cells. The cells were exposed to 1% O₂ with or without rhMIF treatment. Cells were harvested for immunoblot assay for MIF HIF-1α protein. (E) MCF-7 cells were transfected with HRE reporter p2.1 and pRL-SV40 encoding <i>Renilla</i> luciferase. Cells were treated with the indicated dose of rhMIF and exposed to 20% or 1% O₂ conditions for 18 h and then harvested. Results shown represent mean±S.D. of three independent transfections. *; <i>p</i><0.05 compared to respective controls (ANOVA).</p

Propofol induces a metabolic switch to glycolysis and cell death in a mitochondrial electron transport chain-dependent manner

<div><p>The intravenous anesthetic propofol (2,6-diisopropylphenol) has been used for the induction and maintenance of anesthesia and sedation in critical patient care. However, the rare but severe complication propofol infusion syndrome (PRIS) can occur, especially in patients receiving high doses of propofol for prolonged periods. <i>In vivo</i> and <i>in vitro</i> evidence suggests that the propofol toxicity is related to the impaired mitochondrial function. However, underlying molecular mechanisms remain unknown. Therefore, we investigated effects of propofol on cell metabolism and death using a series of established cell lines of various origins, including neurons, myocytes, and trans-mitochondrial cybrids, with defined mitochondrial DNA deficits. We demonstrated that supraclinical concentrations of propofol in not less than 50 μM disturbed the mitochondrial function and induced a metabolic switch, from oxidative phosphorylation to glycolysis, by targeting mitochondrial complexes I, II and III. This disturbance in mitochondrial electron transport caused the generation of reactive oxygen species, resulting in apoptosis. We also found that a predisposition to mitochondrial dysfunction, caused by a genetic mutation or pharmacological suppression of the electron transport chain by biguanides such as metformin and phenformin, promoted propofol-induced caspase activation and cell death induced by clinical relevant concentrations of propofol in not more than 25 μM. With further experiments with appropriate <i>in vivo</i> model, it is possible that the processes to constitute the molecular basis of PRIS are identified.</p></div

Propofol induced cell death and decreased mitochondrial membrane potential in a concentration- and time-dependent manner.

<p>(A) SH-SY5Y cells were exposed to the indicated concentrations (12.5, 25, 50, 100, or 150 μM) of propofol for 6 h and 12 h. LDH release was assayed in culture supernatants (n = 3). Treatment with lysis buffer served as a control. (B) Average mitochondrial membrane potential (ΔΨm) of untreated SH-SY5Y cells and SH-SY5Y cells treated with the indicated concentrations (25, 50, or 100 μM) of propofol (n = 3) for 6 h. Values indicate the ratio [Q2/(Q2 + Q4)] of green JC-1 monomers (527 nm emission) to red aggregates (590 nm emission). Data are expressed as the mean ± SD. Differences between treatment groups were evaluated by two-way ANOVA, followed by Tukey's multiple comparison test (A), or by one-way ANOVA, followed by Tukey's multiple comparison test (B). *<i>p</i> < 0.05 compared to the control cell population (incubation for 0 h, no treatment).</p

Oxygen metabolism and ROS generation in SH-SY5Y cells and P21 cells treated with propofol.

<p>OCR (A, C, and E) and ECAR (B, D, and F) in SH-SY5Y cells or P29 cells exposed to the indicated concentrations of propofol (12.5, 25, 50, or 100 μM) for 6 h (A, B, E and F, respectively) or 0, 3, 6, or 12 h (C and D). Data presented are expressed as the mean ± SD. Differences between treatment groups were evaluated by one-way ANOVA, followed by Dunnett’s multiple comparison test (A, B, E and F), or by two-way ANOVA, followed by Dunnett’s multiple comparison test (C and D). (G) ROS production was measured in SH-SY5Y cells exposed to 25, 50, or 100 μM propofol (n = 3) for 3 h or 6 h. (H) SH-SY5Y cells were exposed to the indicated concentrations (50 or 100 μM) of propofol for 6 h with or without treatment with 10 mM <i>N</i>-acetylcysteine. Cells were harvested, and percentages of cell death were measured by flow cytometry. MFI: median fluorescence intensity; NAC: <i>N</i>-acetylcysteine. Data presented are expressed as the mean ± SD. Differences between treatment groups were evaluated by two-way ANOVA, followed by Tukey's multiple comparison test (G), or by one-way ANOVA, followed by Tukey's multiple comparison test (H). *<i>p</i> < 0.05 compared to the control cell population.</p

Synergistic effects of propofol and metformin on caspase activity and cell death.

<p>(A) OCR and (B) ECAR of SH-SY5Y cells exposed to the indicated concentrations of metformin (2.5, 5, 10, or 20 mM) with or without propofol for 6 h. (C) SH-SY5Y cells were exposed to 25 μM propofol with or without 5 mM metformin for 6 h, and ROS production was determined (n = 3). (D and E) SH-SY5Y cells were exposed to the indicated concentrations (12.5, 25, 50, or 100 μM) of propofol with or without 5 mM metformin for 6 h. (D) Cells were harvested, and percentages of cell death were measured by flow cytometry. The ratio of PI-positive and/or annexin V-positive cells [(Q1 + Q2 + Q4)/(Q1 + Q2 + Q3 + Q4)] was used to calculate the percentage of dead cells (n = 3) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192796#pone.0192796.s004" target="_blank">S1 Fig</a>). (E) Caspase-3/7 activity in each treatment group (n = 3). Data presented in (A–E) are expressed as the mean ± SD. Differences between treatment groups were evaluated by one-way ANOVA, followed by Dunnett’s multiple comparison test (A, B, and C), or by two-way ANOVA, followed by Tukey's multiple comparison test (D and E). *<i>p</i> < 0.05 compared to the control cell population; #<i>p</i> < 0.05 compared to the indicated experimental groups.</p