(-)-Epigallocatechin-3-Gallate Induces Non-Apoptotic Cell Death in Human Cancer Cells via ROS-Mediated Lysosomal Membrane Permeabilization

10.1371/journal.pone.0046749PLoS ONE710

Oxidative Stress-Mediated Skeletal Muscle Degeneration: Molecules, Mechanisms, and Therapies

Oxidative stress is a loss of balance between the production of reactive oxygen species during cellular metabolism and the mechanisms that clear these species to maintain cellular redox homeostasis. Increased oxidative stress has been associated with muscular dystrophy, and many studies have proposed mechanisms that bridge these two pathological conditions at the molecular level. In this review, the evidence indicating a causal role of oxidative stress in the pathogenesis of various muscular dystrophies is revisited. In particular, the mediation of cellular redox status in dystrophic muscle by NF-κB pathway, autophagy, telomere shortening, and epigenetic regulation are discussed. Lastly, the current stance of targeting these pathways using antioxidant therapies in preclinical and clinical trials is examined

Experimental and Model Comparisons of H2O2 Assisted UV Photodegradation of Microcystin-LR in Simulated Drinking Water

The degradation of Microcystin-LR (MC-LR) in water by hydrogen peroxide assisted ultraviolet (UV/H2O2) process was investigated in this paper. The UV/H2O2 process appeared to be effective in removal of the MC-LR. MC-LR decomposition was primarily ascribed to production of strong and nonselective oxidant-hydroxyl radicals within the system. The intensity of UV radiation, initial concentration of MC-LR, MC-LR purity, dosages of H2O2, the initial solution pH, and anions present in water, to some extent, influenced the degradation rate of MC-LR. A modified pseudo-first-order kinetic model was developed to predict the removal efficiency under different experimental conditions

Discovery of New Eunicellin-Based Diterpenoids from a Formosan Soft Coral Cladiella sp.

A new eunicellin diterpenoid, cladieunicellin I (1), and a new natural eunicellin, litophynin I diacetate (2), were isolated from a Formosan soft coral identified as Cladiella sp. The structures of eunicellins 1 and 2 were elucidated by spectroscopic methods and by comparison of the spectral data with those of related analogues. Eunicellin 1 exhibited significant cytotoxicity toward the DLD-1 human colorectal adenocarcinoma cells

ROS mediates LMP and cell death induced by EGCG.

<p>(A) EGCG induces intracellular ROS production. HepG2 cells were treated with EGCG (60 µM) for 6 h in full medium or in serum-free medium Treatment with H₂O₂ (200 µM) for 3 h was used as a positive control. The intracellular ROS was detected by CM-H₂DCFDA and analyzed under a fluorescence microscope. (B) NAC prevents ROS formation induced by EGCG in serum-free medium. Cells were treated with EGCG (60 µM×6 h) in the absence or presence of N-acetylcysteine (NAC, 5 mM). (C) Protection by NAC of EGCG-induced cell death. HepG2 cells were treated by EGCG (60 µM) or H₂O₂ (200 µM) as shown for 12 h in the absence or presence of 5 mM NAC. The cell viability was determined by Hoechst-PI double staining (n = 3, mean ± SD). **<i>P</i><0.005 in comparison to the group without NAC (Student's <i>t</i>-test). (D) NAC prevents cathepsin D translocation caused by EGCG. HepG2 cells were treated with EGCG (60 µM×6 h) with or without 5 mM NAC. Both the lysosomal and cytosolic fractions were analysed by western blot. (E) NAC prevents EGCG-induced cytosolic acidification. HepG2 cells were treated with EGCG (60 µM×6 h) or H₂O₂ (200 µM×3 h) with or without 5 mM NAC. Cells were then stained with AO for 30 min and analyzed by confocal (scale bar: 20 µm). (F) CQ fails to affect ROS production induced by EGCG. HepG2 cells were cultured in serum-free medium for 1 h, then added 60 µM EGCG, with or without the presence of 20 µM CQ for 6 h. (G) Illustration for the mechanisms underlying EGCG-mediated caspase-independent cell death, involving ROS and LMP.</p

EGCG induces LMP.

<p>(A) Effect of EGCG on intracellular acidic compartments. HepG2 cells were with EGCG (60 µM) for indicated durations or with Baf A1 (50 nM) for 6 h. The acidic compartments were labeled by 5 nM Lyo-Tracker Red and examined by confocal (scale bar: 20 µm). (B) EGCG induces the leakage of cathepsins from lysosome to cytosol. Cell fractionation was performed to separate lysosomal and cytosolic fractions in HepG2 treated with 60 µM EGCG in serum-free medium as indicated. Cathepsin D was detected by western blotting in the different fractions and whole cell lysates. LAMP-1 was used as a marker for lysosome. (C) EGCG causes lysosomal neutralization and cytosolic acidification. HepG2 cells were treated with 60 µM EGCG as indicated in serum-free medium, followed by staining with 5 µg/ml acridine orange (AO) for 30 min and analyzed by confocal (scale bar: 20 µm).</p

Serum starvation enhances EGCG-induced cell death independent of caspase.

<p>(A) Serum deprivation promotes EGCG-induced cell death in a concentration-dependent and time-course manner. HepG2 cells were treated with different doses of EGCG in full or serum-free medium for 12 h (left panel) or with 60 µM EGCG for different time as indicated (right panel). The cell viability was determined by Hoechst-PI double staining (n = 3, mean ± SD). (B) Representative pictures of Hoechst-PI double staining. HepG2 cells were cultured in full medium (as a control); treated with 60 µM EGCG for 12 h in serum-free medium; or incubated with 20 ng/ml TNFα and 10 µg/ml CHX for 12 h in full medium (as a positive control for apoptosis). (C) EGCG induces caspase-independent cell death. HepG2 cells were treated with EGCG (60 µM×24 h) or in the absence or presence of 40 µM z-VAD-fmk. The co-treatment with TNFα (20 ng/ml) and CHX (10 µg/ml) for 12 h was used as a positive control. Cell viability was determined as described in Panel A. **<i>p</i><0.005 comparing to the group without z-VAD (Student's <i>t</i>-test, n = 3). (D) No caspase-3 activation and PARP cleavage cause by EGCG-induced cell death. Cells were treated with EGCG or TNF/CHX as described in panel C, and cell lysates were collected and subject to western blot.</p

EGCG induces cytosolic vacuolization.

<p>(A) Morphological alterations of EGCG-treated cells in serum-free medium were analyzed using light microscopy. Representative pictures of HepG2 cell treated with EGCG at indicated concentrations for 12 h (upper panel) or with 60 µM EGCG for indicated time courses (lower panel) are shown (scale bar: 50 µm). (B) HepG2 cells were treated with EGCG (60 or 240 µM) for 12 h. The cells were then fixed and stained by hematoxylin, then analyzed by light microscopy (scale bar: 30 µm). (C) The vacuole contents are not lipid droplets. HepG2 cells were EGCG (60 µM) for 12 h. Cultured cells in full medium for 12 h were used as a negative control, and cells treated with 1 mM oleate acid (OA) in DMEM medium containing 1%BSA for 12 h were used as the positive control. The cells were fixed and stained with 0.5% Oil Red O and hematoxylin (scale bar: 30 µm). (D) The vacuoles are of lysosome origin. Immunofluorescence staining of LAMP-1 was performed in MEF cells after treatment with or without EGCG (60 µM) for 9 h (scale bar: 10 µm).</p