Role of oxidative stress in lysosomal membrane permeabilization and apoptosis induced by gentamicin, an aminoglycoside antibiotic

Gentamicin, an aminoglycoside antibiotic used to treat severe bacterial infections, may cause acute renal failure. At therapeutic concentrations, gentamicin accumulates in lysosomes and induces apoptosis in kidney proximal tubular cells. In gentamicin-treated renal LLC-PK1 cells, acridine orange release from lysosomes, previously interpreted as lysosomal membrane permeabilization, precedes the apoptotic cascade that develops during incubation with gentamicin. However, the link between gentamicin lysosomal accumulation and apoptosis remains unclear. We here examined if reactive oxygen species (ROS) production could account for gentamicin-induced acridine orange release and apoptosis, and the implication of iron in these events. We found that gentamicin induced ROS production prior to, and at lower drug concentrations than required for, acridine orange release and apoptosis. ROS antioxidant or scavenger, catalase, and N-acetylcysteine largely prevented these events. Vital confocal imaging revealed that gentamicin-induced ROS production occurs in lysosomes. Deferoxamine, an iron chelator, which is endocytosed and accumulates in lysosomes, largely prevented gentamicin-induced ROS production as well as apoptosis. Direct evidence for gentamicin-induced permeabilization of lysosomal membrane was provided by showing the release into the cytosol of Lucifer yellow, a membrane-impermeant endocytic tracer with a comparable molecular weight as gentamicin. Altogether, our data demonstrate a key role of lysosomal iron and early ROS production in gentamicin-induced lysosomal membrane permeabilization and apoptosis.     

Suberoylanilide hydroxamic acid induces ROS-mediated cleavage of HSP90 in leukemia cells

Heat shock protein 90 (HSP90) is a molecular chaperone that supports stability of client proteins. We found that HSP90 was cleaved to 55 kDa protein after treatment with histone deacetylase (HDAC) inhibitors including suberoylanilide hydroxamic acid (SAHA) in several leukemia cell lines. We further analyzed molecular changes induced by SAHA in K562 cells. The SAHA-induced cleavage of HSP90 was blocked by a pan-caspase inhibitor, z-VAD-fmk, implying that the process is dependent on caspase activity. However, the experiments using antagonistic and agonistic Fas antibodies revealed that the cleavage of HSP90 was not dependent on Fas signaling. SAHA induced generation of reactive oxygen species (ROS), and the cleavage of HSP90 was blocked by a ROS scavenger N-acetylcystein (NAC). We also confirmed that hydrogen peroxide (H₂O₂) induced cleavage of HSP90 in a similar manner. Caspase 2, 3, 4, 6, 8, and 10 were activated by treatment with SAHA, and the activities were reduced by the pretreatment of NAC. Treatment of the cells with caspase 10 inhihitor, but not other inhibitors of caspases activated by SAHA, prevented cleavage of HSP90 by SAHA. SAHA-induced ROS generation and HSP90 cleavage were dependent on newly synthesized unknown proteins. Taken together, our results suggest that the cleavage of HSP90 by SAHA is mediated by ROS generation and caspase 10 activation. HSP90 cleavage may provide an additional mechanism involved in anti-cancer effects of HDAC inhibitors.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-014-0533-4) contains supplementary material, which is available to authorized users.     

Mitochondrial membrane permeabilization: the sine qua non for cell death

Mitochondria are essential for maintaining cell life but they also play a role in regulating cell death, which occurs when their membranes become permeabilized. Mitochondria possess two distinct membrane systems including an outer membrane in close communication with the cytosol and an inner membrane involved in energy transduction. Outer membrane permeabilization is regulated by Bcl-2 family proteins, which control the release of proteins from the mitochondrial intermembrane space; these proteins then activate apoptosis. Inner membrane permeabilization is regulated by the mitochondrial permeability transition (MPT), which is activated by calcium and oxidative stress and leads to bioenergetic failure and necrosis. The purpose of this review is to discuss the biochemical mechanisms regulating mitochondrial membrane permeabilization; this is crucial to our understanding of the role of cell death in diseases such as cancer and the neurodegenerative diseases.     

Facile synthetic nano-curcumin encapsulated Bio-fabricated nanoparticles induces ROS-mediated apoptosis and migration blocking of human lung cancer cells

Lung cancer is one of the major life-threatening cancers, with a higher mortality rate and morbidity. Curcumin acts as a potential anticancer agent but it shows less aqueous solubility so its clinical application is limited. In this present study, we developed curcumin-surface decorated Zinc Oxide nanoparticles ZnO@Cur to enhance its aqueous formulation and improve the anti-cancer activity of curcumin. The synthesized ZnO@Cur confirmed by various spectroscopies (UV–vis and fluorescence) and electroscopic techniques (Powder XRD, SEM, TEM, and DLS). ZnO@Cur dramatically suppressed the proliferation of A549 and HEL-299 cells and showed low toxicity in MTT assays. Compared with free ZnO and curcumin, ZnO@Cur significantly inhibited the migration ability of A549 cells. Moreover, the induction of apoptosis in A549 cells by ZnO@Cur was resulted by AO-EB staining. ZnO@Cur activated to promote intracellular ROS overproduction and induced apoptosis. ZnO@Cur shows excellent biocompatibility compared to free ZnO and Curcumin, the present study explained that ZnO@Cur as a safe and hopeful strategy for chemotherapeutics of lung cancer therapy and deserve for further clinical evaluations.     

Ergosterol depletion under bifonazole treatment induces cell membrane damage and triggers a ROS-mediated mitochondrial apoptosis in Penicillium expansum

Penicillium expansum is the causal agent of blue mold in harvested fruits and vegetables during storage and distribution, causing serious economic loss. In this study we seek the action modes of bifonazole against this pathogen. Bifonazole exhibited strong antifungal activity against P. expansum by inhibiting ergosterol synthesis. The ergosterol depletion caused damage to the cell structure and especially cell membrane integrity as observed by SEM and TEM. With increased unsaturated fatty acids contents, the cell membrane viscosity decreases and can no longer effectively maintain the cytoplasm, which ultimately decreases extracellular conductivity, changes intracellular pH and ion homeostasis. Exposure of hyphal cells to bifonazole shows that mitochondrial respiration is inhibited and reactive oxygen species (ROS) levels–including H₂O₂ and malondialdehyde (MDA) – are significantly increased. The functional impairment of mitochondria and cell membrane eventually cause cell death through intrinsic apoptosis and necroptosis.     

ω-Hydroxyundec-9-enoic acid induces apoptosis through ROS-mediated endoplasmic reticulum stress in non-small cell lung cancer cells

ω-Hydroxyundec-9-enoic acid (ω-HUA), a hydroxyl unsaturated fatty acid derivative, is involved in the antifungal activity of wild rice (Oryza officinalis). Here, we investigated the anti-cancer activity of ω-HUA on a non-small cell lung cancer (NSCLC) cell line. ω-HUA increased apoptosis and induced cleavages of caspase-6, caspase-9, and poly (ADP-ribose) polymerase (PARP). ω-HUA treatment significantly induced endoplasmic reticulum (ER) stress response. Suppression of CHOP expression and inhibiting ER stress by 4-phenylbutyrate (4-PBA) significantly attenuated the ω-HUA treatment-induced activation of caspase-6, caspase-9, and PARP, and subsequent apoptotic cell death, indicating a role for ER stress in ω-HUA-induced apoptosis. In addition, cells subjected to ω-HUA exhibited significantly increased quantity of reactive oxygen species (ROS), and the ROS scavenger N-acetyl-l-cysteine (NAC) inhibited ω-HUA-induced apoptotic cell death and ER stress signals, indicating a role for ROS in ER stress-mediated apoptosis in ω-HUA-treated cells. Taken together, these results suggest that sequential ROS generation and ER stress activation are critical in ω-HUA treatment-induced apoptosis and that ω-HUA represents a promising candidate for NSCLC treatment.     

Surfactant protein SP-B induces ordering at the surface of model membrane bilayers

The effects of bovine pulmonary surfactant-associated protein B (SP-B) on molecular packing of model membrane lipids (7:1 DPPC/DPPG) were studied by fluorescence anisotropy. The bilayer surface was markedly ordered by SP-B below the gel to fluid phase transition temperature (Tc) while it was only slightly ordered above this temperature as indicated by surface-sensitive probes 6-NBD-PC and 6-NBD-PG. The effects of SP-B on fluorescence anisotropy were concentration dependent, reaching maximal activity at 1-2% protein to phospholipid by weight. Anisotropy measurements of interior-selective fluorescent probes (cis-parinaric acid and DPH) imply that addition of SP-B into the phospholipid shifted the Tc of the model membrane but did not alter lipid order at the membrane interior. Since fluorescence anisotropy studies with trans-parinaric acid, an interior-sensitive probe with high affinity for gel-phase lipids, did not detect any changes in lipid packing or Tc, it is likely that SP-B resides primarily in fluid-phase domains. Fluorescence lifetime measurements indicated that two conformers of the NBD-PC probe exist in this DPPC/DPPG model membrane system. Fluorescence intensity measurements generated with NBD-PC and NBD-PG, in conjunction with information from lifetime measurements, support the concept that SP-B increases the distribution of the short-lifetime conformer in the gel phase. In addition, the anisotropy and intensity profiles of NBD-PG in the model membrane indicate that bovine SP-B interacts selectively with phosphatidylglycerol.     

Alamethicin or detergent permeabilization of the cell membrane as a tool for adenylate cyclase determination

Treatment of mouse lymphocytes with very low concentrations of alamethicin or Lubrol PX induces spontaneous permeabilization of the plasma membrane to ATP and allows determination of adenylate cyclase activity in whole cells. The permeabilized cells retain responsiveness to hormones (isoproterenol, adenosine analogs) and to fluoride. The main advantage of this new method is that it does not require any homogenization step, and thus adenylate cyclase activities can be accurately and reproducibly measured with very low amounts of cells. It should be especially useful for the study of purified lymphocyte subpopulations.     

Methods for the assessment of mitochondrial membrane permeabilization in apoptosis

Mitochondrial membrane permeabilization (MMP) is considered as the “point-of-no-return” in numerous models of programmed cell death. Indeed, mitochondria determine the intrinsic pathway of apoptosis, and play a major role in the extrinsic route as well. MMP affects the inner and outer mitochondrial membranes (IM and OM, respectively) to a variable degree. OM permeabilization culminates in the release of proteins that normally are confined in the mitochondrial intermembrane space (IMS), including caspase activators (e.g. cytochrome c) and caspase-independent death effectors (e.g. apoptosis-inducing factor). Partial IM permeabilization disrupts mitochondrial ion and volume homeostasis and dissipates the mitochondrial transmembrane potential (ΔΨ_m). The assessment of early mitochondrial alterations allows for the identification of cells that are committed to die but have not displayed yet the apoptotic phenotype. Several techniques to measure MMP by cytofluorometry and fluorescence microscopy have been developed. Here, we summarize the currently available methods for the detection of MMP, and provide a comparative analysis of these techniques.     

Roles of integral protein in membrane permeabilization by amphidinols

Amphidinols (AMs) are a group of dinoflagellate metabolites with potent antifungal activity. As is the case with polyene macrolide antibiotics, the mode of action of AMs is accounted for by direct interaction with lipid bilayers, which leads to formation of pores or lesions in biomembranes. However, it was revealed that AMs induce hemolysis with significantly lower concentrations than those necessary to permeabilize artificial liposomes, suggesting that a certain factor(s) in erythrocyte membrane potentiates AM activity. Glycophorin A (GpA), a major erythrocyte protein, was chosen as a model protein to investigate interaction between peptides and AMs such as AM2, AM3 and AM6 by using SDS-PAGE, surface plasmon resonance, and fluorescent-dye leakages from GpA-reconstituted liposomes. The results unambiguously demonstrated that AMs have an affinity to the transmembrane domain of GpA, and their membrane-permeabilizing activity is significantly potentiated by GpA. Surface plasmon resonance experiments revealed that their interaction has a dissociation constant of the order of 10 μM, which is significantly larger than efficacious concentrations of hemolysis by AMs. These results imply that the potentiation action by GpA or membrane integral peptides may be due to a higher affinity of AMs to protein-containing membranes than that to pure lipid bilayers.     

VDAC-dependent permeabilization of the outer mitochondrial membrane by superoxide induces rapid and massive cytochrome c release.

Enhanced formation of reactive oxygen species (ROS), superoxide (O2*-), and hydrogen peroxide (H2O2) may result in either apoptosis or other forms of cell death. Here, we studied the mechanisms underlying activation of the apoptotic machinery by ROS. Exposure of permeabilized HepG2 cells to O2*- elicited rapid and massive cytochrome c release (CCR), whereas H2O2 failed to induce any release. Both O2*- and H2O2 promoted activation of the mitochondrial permeability transition pore by Ca2+, but Ca2+-dependent pore opening was not required for O2*--induced CCR. Furthermore, O2*- alone evoked CCR without damage of the inner mitochondrial membrane barrier, as mitochondrial membrane potential was sustained in the presence of extramitochondrial ATP. Strikingly, pretreatment of the cells with drugs or an antibody, which block the voltage-dependent anion channel (VDAC), prevented O2*--induced CCR. Furthermore, VDAC-reconstituted liposomes permeated cytochrome c after O2*- exposure, and this release was prevented by VDAC blocker. The proapoptotic protein, Bak, was not detected in HepG2 cells and O2*--induced CCR did not depend on Bax translocation to mitochondria. O2*--induced CCR was followed by caspase activation and execution of apoptosis. Thus, O2*- triggers apoptosis via VDAC-dependent permeabilization of the mitochondrial outer membrane without apparent contribution of proapoptotic Bcl-2 family proteins.     

Productive HIV-1 infection of primary CD4+ T cells induces mitochondrial membrane permeabilization leading to a caspase-independent cell death.

We have explored in vitro the mechanism by which human immunodeficiency virus, type 1 (HIV-1) induces cell death of primary CD4+ T cells in conditions of productive infection. Although HIV-1 infection primed phytohemagglutinin-activated CD4+ T cells for death induced by anti-CD95 antibody, T cell death was not prevented by a CD95-Fc decoy receptor, nor by decoy receptors of other members of the TNFR family (TNFR1/R2, TRAILR1/R2/OPG, TRAMP) or by various blocking antibodies, suggesting that triggering of death receptors by their cognate ligands is not involved in HIV-induced CD4 T cell death. HIV-1 induced CD4 T cell shrinkage, cell surface exposure of phosphatidylserine, loss of mitochondrial membrane potential (Deltapsim), and mitochondrial release of cytochrome c and apoptosis-inducing factor. A typical apoptotic phenotype (nuclear chromatin condensation and fragmentation) only occurred in around half of the dying cells. Treatment with benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a broad spectrum caspase inhibitor, prevented nuclear chromatin condensation and fragmentation in HIV-infected CD4+ T cells and in a cell-free system (in which nuclei were incubated with cytoplasmic extracts from the HIV-infected CD4+ T cells). Nevertheless, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone did not prevent mitochondrial membrane potential loss and cell death, suggesting that caspases are dispensable for HIV-mediated cell death. Our findings suggest a major role of the mitochondria in the process of CD4 T cell death induced by HIV, in which targeting of Bax to the mitochondria may be involved.     

Cd2+ versus Ca2+-produced mitochondrial membrane permeabilization: a proposed direct participation of respiratory complexes I and III

A comparison of Cd²⁺ and Ca²⁺ effects on in vitro rat liver mitochondria function and a further study of their interaction were conducted. Similarity and distinction in action of rotenone, oligomycin, N-ethylmaleimide, dithiothreitol, catalase, dibucaine, ruthenium red, cyclosporin A (CsA), and ADP on Cd²⁺ and/or Ca²⁺-induced mitochondrial dysfunction were revealed. We found that rotenone exerted a strong protective action both against Ca²⁺ and Cd²⁺-produced mitochondrial membrane permeabilization (MMP). In contrast to Ca²⁺, catalase and dibucaine did not influence on main Cd²⁺ effects. In NH₄NO₃ medium N-ethylmaleimide (NEM) at low concentrations increased markedly Cd²⁺-produced swelling of non-energized mitochondria, whereas it exhibited a partial reversal effect following energization. In sucrose medium low [NEM] did not change Cd²⁺-produced mitochondrial swelling. High [NEM] promoted synergistic increase of the Cd²⁺-produced swelling in NH₄NO₃ medium; all above effects were reversed (and prevented) by dithiothreitol, DTT. We shown also that when exogenous Ca²⁺ and P_i were simultaneously present in NH₄NO₃ medium, DTT reversed only partially Cd²⁺-produced swelling of succinate plus rotenone-energized mitochondria, while DTT recovery action was complete when either Ca²⁺ or P_i were separately administered to the Cd²⁺-treated mitochondria. Besides, DTT added following a low Cd²⁺ pulse in KCl medium containing exogenous Ca²⁺ induced a substantial enhancing of sustained Cd²⁺ stimulation of mitochondrial basal respiration and the stimulation was CsA-sensitive, while the activation promoted by low [Cd²⁺] alone was totally eliminated by DTT supplement. We observed the similar respiratory activation earlier when high concentrations of Cd²⁺ in the absence of added Ca²⁺ were used but it was completely CsA-insensitive. A possible involvement of respiratory chain components, namely complex I (P-site) and complex III (S-site) in Cd²⁺and/or Ca²⁺-produced MMP was discussed.     

Permeability and the hidden area of lipid bilayers

The passive water permeability of a lipid vesicle membrane was studied, related to the hydrostatic (not osmotic) pressure difference between the inner and the outer side of the vesicle in a water environment without additives. Each pressure difference was created by sucking a vesicle into a micropipette at a given sucking pressure. The part of the membrane sucked into the micropipette (the projection length) was measured as a function of time. The time dependence can be divided into two intervals. We put forward the idea that smoothing of membrane defects, accompanied by an increase of the membrane area, takes place during the initial time interval, which results in a faster increase of the projection length. In the second time interval the volume of the vesicle decreases due to the permeability of its membrane and the increase of the projection length is slower. The hidden area and the water permeability of a typical lipid bilayer were estimated. The measured permeability, conjugated to the hydrostatic pressure difference, is an order of magnitude higher than the known value of the permeability, conjugated to the osmotic pressure difference. A hypothesis, based on pore formation, is proposed as an explanation of this experimental result.     

Menadione (vitamin K) enhances the antibiotic activity of drugs by cell membrane permeabilization mechanism

Menadione, vitamin K3, belongs to the class of lipid-soluble vitamins and lipophilic substances as menadione cause disturbances in the bacterial membrane, resulting in damage to the fundamental elements for the integrity of the membrane, thus allowing increased permeability. Accordingly, the aim of this study was to evaluate in vitro the antibiotic-modifying activity of menadione in multiresistant strains of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, with a gradual increase in its subinhibitory concentration. In addition, menadione was compared with cholesterol and ergosterol for similarity in mechanism of drug modulatory action. Antibiotic-modifying activity and antibacterial effect were determined by the broth microdilution assay. Menadione, cholesterol and ergosterol showed modulatory activity at clinically relevant concentrations, characterizing them as modifiers of bacterial drug resistance, since they lowered the MIC of the antibiotics tested. This is the first report of the antibacterial activity of menadione and its potentiation of aminoglycosides against multiresistant bacteria.     

Unsteady-state operation of continuous fermentor for enhancement of cell mass production

The transient behavior of continuous fermentation is studied concentrating on the time scale intrinsic to the system. The time scale is the time required for the fermentorto reach a stable steady state after the disturbance of cell mass is introduced. When the cell concentration is disturbed from the steady-state value, in particular, at the dilution rate near washout, the transient period becomes extended significantly, and the steady state is resumed sluggishly. This sluggish transient behavior could be turned to an advantage for enhancing the cell mass output rate. The proposed transient operation is a continuous fermentation whereby a positive disturbance in the cell mass is introduced, so that the cell concentration is higher than the steady-state value for an extended transient period. It is shown that a significantly higher cell mass production than that from the steady-state continuous fermentation can be achieved. Simple experiments were performed to demonstrate the improvement of cell (Candida utilis) productivity.     

The aromatic domain of the coronavirus class I viral fusion protein induces membrane permeabilization: putative role during viral entry

Coronavirus (CoV) entry is mediated by the viral spike (S) glycoprotein, a class I viral fusion protein. During viral and target cell membrane fusion, the heptad repeat (HR) regions of the S2 subunit assume a trimer-of-hairpins structure, positioning the fusion peptide in close proximity to the C-terminal region of the ectodomain. The formation of this structure appears to drive apposition and subsequent fusion of viral and target cell membranes; however, the exact mechanism is unclear. Here, we characterize an aromatic amino acid rich region within the ectodomain of the S2 subunit that both partitions into lipid membranes and has the capacity to perturb lipid vesicle integrity. Circular dichroism analysis indicated that peptides analogous to the aromatic domains of the severe acute respiratory syndrome (SARS)-CoV, mouse hepatitis virus (MHV) and the human CoV OC43 S2 subunits, did not have a propensity for a defined secondary structure. These peptides strongly partitioned into lipid membranes and induced lipid vesicle permeabilization at peptide/lipid ratios of 1:100 in two independent leakage assays. Thus, partitioning of the peptides into the lipid interface is sufficient to disorganize membrane integrity. Our study of the S2 aromatic domain of three CoVs provides supportive evidence for a functional role of this region. We propose that, when aligned with the fusion peptide and transmembrane domains during membrane apposition, the aromatic domain of the CoV S protein functions to perturb the target cell membrane and provides a continuous track of hydrophobic surface, resulting in lipid-membrane fusion and subsequent viral nucleocapsid entry.     

Development of a microfluidic device for determination of cell osmotic behavior and membrane transport properties

An understanding of cell osmotic behavior and membrane transport properties is indispensable for cryobiology research and development of cell-type-specific, optimal cryopreservation conditions. A microfluidic perfusion system is developed here to measure the kinetic changes of cell volume under various extracellular conditions, in order to determine cell osmotic behavior and membrane transport properties. The system is fabricated using soft lithography and is comprised of microfluidic channels and a perfusion chamber for trapping cells. During experiments, rat basophilic leukemia (RBL-1 line) cells were injected into the inlet of the device, allowed to flow downstream, and were trapped within a perfusion chamber. The fluid continues to flow to the outlet due to suction produced by a Hamilton Syringe. Two sets of experiments have been performed: the cells were perfused by (1) hypertonic solutions with different concentrations of non-permeating solutes and (2) solutions containing a permeating cryoprotective agent (CPA), dimethylsulfoxide (Me₂SO), plus non-permeating solute (sodium chloride (NaCl)), respectively. From experiment (1), cell osmotically inactive volume (V_b) and the permeability coefficient of water (L_p) for RBL cells are determined to be 41% [n = 18, correlation coefficient (r²) of 0.903] of original/isotonic volume, and 0.32 ± 0.05 μm/min/atm (n = 8, r² > 0.963), respectively, for room temperature (22 °C). From experiment (2), the permeability coefficient of water (L_p) and of Me₂SO (P_s) for RBL cells are 0.38 ± 0.09 μm/min/atm and (0.49 ± 0.13) × 10⁻³ cm/min (n = 5, r² > 0.86), respectively. We conclude that this device enables us to: (1) readily monitor the changes of extracellular conditions by perfusing single or a group of cells with prepared media; (2) confine cells (or a cell) within a monolayer chamber, which prevents imaging ambiguity, such as cells overlapping or moving out of the focus plane; (3) study individual cell osmotic response and determine cell membrane transport properties; and (4) reduce labor requirements for its disposability and ensure low manufacturing costs.     

Physical properties of membrane fractions isolated from human platelets: implications for chilling induced platelet activation

In previous studies, it has been suggested that chilling induced activation of human platelets is related to a lipid phase transition seen in membrane lipids. Those studies showed a single, surprisingly cooperative transition in human platelets, as determined by Fourier transform infrared (FTIR) spectroscopy, findings that are confirmed here with calorimetric measurements. Such transitions have now been studied in membrane fractions obtained from the platelets and it is reported that all fractions and purified phospholipids show similar transitions. In order to obtain these data it was necessary to develop means for separating these fractions. Therefore, a novel method for isolation and separation of dense tubular system (DTS) and plasma membranes in human platelets is described here. Lipid analysis showed that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the dominant phospholipids in both fractions, whereas cholesterol and sphingomyelin (SM) were predominantly located in the plasma membranes. Thermotropic phase transitions in the two membrane fractions, determined by differential scanning calorimetry (DSC) and FTIR spectroscopy were found to occur at about 15 degrees C, similar to the Tm of intact human platelets. These data are discussed in relation to the role of the DTS and plasma membranes in the cold-induced activation of human platelets.     

BIX-01294 induces autophagy-associated cell death via EHMT2/G9a dysfunction and intracellular reactive oxygen species production

We screened a chemical library in MCF-7 cells stably expressing green fluorescent protein (GFP)-conjugated microtubule-associated protein 1 light chain 3 (LC3) (GFP-LC3-MCF-7) using cell-based assay, and identified BIX-01294 (BIX), a selective inhibitor of euchromatic histone-lysine N-methyltransferase 2 (EHMT2), as a strong autophagy inducer. BIX enhanced formation of GFP-LC3 puncta, LC3-II, and free GFP, signifying autophagic activation. Inhibition of these phenomena with chloroquine and increasement in punctate dKeima ratio (550/438) signal indicated that BIX activated autophagic flux. BIX-induced cell death was suppressed by the autophagy inhibitor, 3-methyladenine, or siRNA against BECN1 (VPS30/ATG6), ATG5, and ATG7, but not by caspase inhibitors. Moreover, EHMT2 siRNA augmented GFP-LC3 puncta, LC3-II, free GFP, and cell death, implying that inhibition of EHMT2 caused autophagy-mediated cell death. Treatment with EHMT2 siRNA and BIX accumulated intracellular reactive oxygen species (ROS). BIX augmented mitochondrial superoxide via NADPH oxidase activation. In addition, BIX increased hydrogen peroxide and glutathione redox potential in both cytosol and mitochondria. Treatment with N-acetyl-L-cysteine (NAC) or diphenyleneiodonium chloride (DPI) decreased BIX-induced LC3-II, GFP-LC3 puncta, and cell death, indicating that ROS instigated autophagy-dependent cell death triggered by BIX. We observed that BIX potentiated autophagy-dependent and caspase-independent cell death in estrogen receptor (ESR)-negative SKBr3 and ESR-positive MCF-7 breast cancer cells, HCT116 colon cancer cells, and importantly, in primary human breast and colon cancer cells. Together, the results suggest that BIX induces autophagy-dependent cell death via EHMT2 dysfunction and intracellular ROS accumulation in breast and colon cancer cells, therefore EHMT2 inhibition can be an effective therapeutic strategy for cancer treatment.