Eniko Hocsak

Inhibition of cell death by a novel 16.2 kD heat shock protein predominantly via Hsp90 mediated lipid rafts stabilization and Akt activation pathway

AlphaB-crystallin homology, heat stress induction and chaperone activity suggested that a previously encloned gene product is a novel small heat shock protein (Hsp16.2). Suppression of Hsp16.2 by siRNA sensitized cells to hydrogen peroxide or taxol induced cell-death. Over-expressing of Hsp16.2 protected cells against stress stimuli by inhibiting cytochrome c release from the mitochondria, nuclear translocation of AIF and endonuclease G, and caspase 3 activation. Recombinant Hsp16.2 protected mitochondrial membrane potential against calcium induced collapse in vitro indicating that Hsp16.2 stabilizes mitochondrial membrane systems. Hsp16.2 formed self-aggregates and bound to Hsp90. Inhibition of Hsp90 by geldanamycin diminished the cytoprotective effect of Hsp16.2 indicating that this effect was Hsp90-mediated. Hsp16.2 over-expression increased lipid rafts formation as demonstrated by increased cell surface labeling with fluorescent cholera toxin B, and increased Akt phosphorylation. The inhibition of PI-3-kinase—Akt pathway by LY-294002 or wortmannin significantly decreased the protective effect of the Hsp16.2. These data indicate that the over-expression of Hsp16.2 inhibits cell death via the stabilization of mitochondrial membrane system, activation of Hsp90, stabilization of lipid rafts and by the activation of PI-3-kinase—Akt cytoprotective pathway.

Induction of necrotic cell death and mitochondrial permeabilization by heme binding protein 2/SOUL

We found that heme‐binding protein 2/SOUL sensitised NIH3T3 cells to cell death induced by A23187 and etoposide, but it did not affect reactive oxygen species formation. In the presence of sub‐threshold calcium, recombinant SOUL provoked mitochondrial permeability transition (mPT) in vitro that was inhibited by cyclosporine A (CsA). This effect was verified in vivo by monitoring the dissipation of mitochondrial membrane potential. Flow cytometry analysis showed that SOUL promoted necrotic death in A23187 and etoposide treated cells, which effect was prevented by CsA. These data suggest that besides its heme‐binding properties SOUL promotes necrotic cell death by inducing mPT.

TIP47 protects mitochondrial membrane integrity and inhibits oxidative‐stress‐induced cell death

We found that overexpression of tail interacting protein of 47 kDa (TIP47), but not its truncated form (t‐TIP47) protected NIH3T3 cells from hydrogen‐peroxide‐induced cell death, prevented the hydrogen‐peroxide‐induced mitochondrial depolarization determined by 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethyl‐benzimidazolylcarbocyanine iodide (JC1), while suppression of TIP47 in HeLa cells facilitated oxidative‐stress‐induced cell death. TIP47 was located to the cytoplasm of untreated cells, but some was associated to mitochondria in oxidative stress. Recombinant TIP47, but not t‐TIP47 increased the mitochondrial membrane potential (Δψ), and partially prevented Ca2+ induced depolarization. It is assumed that TIP47 can bind to mitochondria in oxidative stress, and inhibit mitochondria mediated cell death by protecting mitochondrial membrane integrity.

Facilitation of Mitochondrial Outer and Inner Membrane Permeabilization and Cell Death in Oxidative Stress by a Novel Bcl-2 Homology 3 Domain Protein

We identified a sequence homologous to the Bcl-2 homology 3 (BH3) domain of Bcl-2 proteins in SOUL. Tissues expressed the protein to different extents. It was predominantly located in the cytoplasm, although a fraction of SOUL was associated with the mitochondria that increased upon oxidative stress. Recombinant SOUL protein facilitated mitochondrial permeability transition and collapse of mitochondrial membrane potential (MMP) and facilitated the release of proapoptotic mitochondrial intermembrane proteins (PMIP) at low calcium and phosphate concentrations in a cyclosporine A-dependent manner in vitro in isolated mitochondria. Suppression of endogenous SOUL by diced small interfering RNA in HeLa cells increased their viability in oxidative stress. Overexpression of SOUL in NIH3T3 cells promoted hydrogen peroxide-induced cell death and stimulated the release of PMIP but did not enhance caspase-3 activation. Despite the release of PMIP, SOUL facilitated predominantly necrotic cell death, as revealed by annexin V and propidium iodide staining. This necrotic death could be the result of SOUL-facilitated collapse of MMP demonstrated by JC-1 fluorescence. Deletion of the putative BH3 domain sequence prevented all of these effects of SOUL. Suppression of cyclophilin D prevented these effects too, indicating that SOUL facilitated mitochondrial permeability transition in vivo. Overexpression of Bcl-2 and Bcl-xL, which can counteract the mitochondria-permeabilizing effect of BH3 domain proteins, also prevented SOUL-facilitated collapse of MMP and cell death. These data indicate that SOUL can be a novel member of the BH3 domain-only proteins that cannot induce cell death alone but can facilitate both outer and inner mitochondrial membrane permeabilization and predominantly necrotic cell death in oxidative stress.

TIP47 confers resistance to taxol-induced cell death by preventing the nuclear translocation of AIF and Endonuclease G

Tail-interacting protein (TIP47, also named PP17) has been implicated in lipid droplet metabolism and in the development of late endosomes, to date however, no data about its possible role in regulating cell death processes has been available. Here, we provide evidence for the role of TIP47 in the regulation of mitochondrial membrane stability and cell death. Overexpression of TIP47 protected NIH3T3 cells from taxol-induced cell death, while suppression of TIP47 by siRNA facilitated cell death. TIP47, but not its truncated form, t-TIP47, decreased taxol-induced cell death as determined by propidium iodide and fluorescent Annexin V staining. Recombinant TIP47, but not t-TIP47, partially prevented taxol-induced depolarization of mitochondria in vitro. Overexpression of TIP47, but not its truncated form, prevented the taxol-induced nuclear and cytoplasmic translocation of AIF and Endonuclease G, as well as the taxol-induced depolarization of mitochondria in NIH3T3 cells. Furthermore, overexpression of TIP47 facilitated Bcl-2 expression and suppressed Bax expression in taxol-treated cells. These data show that besides its previously known functions, TIP47 is involved in the regulation of mitochondria-related cell death by directly stabilizing the mitochondrial membrane system and by favorably affecting the expression of Bcl-2 homologues. Since TIP47 is overexpressed in certain tumors, it is possible that TIP47 contributes to the development of cytostatic resistance.

BGP-15 protects against oxidative stress- or lipopolysaccharide-induced mitochondrial destabilization and reduces mitochondrial production of reactive oxygen species

Reactive oxygen species (ROS) play a critical role in the progression of mitochondria-related diseases. A novel insulin sensitizer drug candidate, BGP-15, has been shown to have protective effects in several oxidative stress-related diseases in animal and human studies. In this study, we investigated whether the protective effects of BGP-15 are predominantly via preserving mitochondrial integrity and reducing mitochondrial ROS production. BGP-15 was found to accumulate in the mitochondria, protect against ROS-induced mitochondrial depolarization and attenuate ROS-induced mitochondrial ROS production in a cell culture model, and also reduced ROS production predominantly at the complex I-III system in isolated mitochondria. At physiologically relevant concentrations, BGP-15 protected against hydrogen peroxide-induced cell death by reducing both apoptosis and necrosis. Additionally, it attenuated bacterial lipopolysaccharide (LPS)-induced collapse of mitochondrial membrane potential and ROS production in LPS-sensitive U-251 glioma cells, suggesting that BGP-15 may have a protective role in inflammatory diseases. However, BGP-15 did not have any antioxidant effects as shown by in vitro chemical and cell culture systems. These data suggest that BGP-15 could be a novel mitochondrial drug candidate for the prevention of ROS-related and inflammatory disease progression.

PARP inhibition protects mitochondria and reduces ROS production via PARP-1-ATF4-MKP-1-MAPK retrograde pathway

Abstract Oxidative stress induces DNA breaks and PARP‐1 activation which initiates mitochondrial reactive oxygen species (ROS) production and cell death through pathways not yet identified. Here, we show the mechanism by which PARP‐1 influences these processes via PARylation of activating transcription factor‐4 (ATF4) responsible for MAP kinase phosphatase‐1 (MKP‐1) expression and thereby regulates MAP kinases. PARP inhibitor, or silencing, of PARP induced MKP‐1 expression by ATF4‐dependent way, and inactivated JNK and p38 MAP kinases. Additionally, it induced ATF4 expression and binding to cAMP‐response element (CRE) leading to MKP‐1 expression and the inactivation of MAP kinases. In contrast, PARP‐1 activation induced the PARylation of ATF4 and reduced its binding to CRE sequence in vitro. CHIP‐qPCR analysis showed that PARP inhibitor increased the ATF4 occupancy at the initiation site of MKP‐1. In oxidative stress, PARP inhibition reduced ROS‐induced cell death, suppressed mitochondrial ROS production and protected mitochondrial membrane potential on an ATF4 and MKP‐1 dependent way. Basically identical results were obtained in WRL‐68, A‐549 and T24/83 human cell lines indicating that the aforementioned mechanism can be universal. Here, we provide the first description of PARP‐1‐ATF4‐MKP‐1‐JNK/p38 MAPK retrograde pathway, which is responsible for the regulation of mitochondrial integrity, ROS production and cell death in oxidative stress, and may represent a new mechanism of PARP in cancer therapy since cancer stem cells development is JNK‐dependent. Graphical abstract Figure. No Caption available. HighlightsATF4 silencing blocked PARP inhibitor‐evoked MKP‐1 upregulation in oxidative stress.Affinity of PARylated ATF‐4 and PARP for CRE decreased and increased, respectively.PARP inhibitor increased ATF4 occupancy of MKP‐1’s initiation site.PARP‐1‐ATF4‐MKP‐1‐JNK/p38 MAPK retrograde pathway mediates the cell death.Relevance in cancer therapy: cancer stem cells development is JNK‐dependent.

Activation of mitochondrial fusion provides a new treatment for mitochondria-related diseases

Graphical abstract Figure. No Caption available. Abstract Mitochondria fragmentation destabilizes mitochondrial membranes, promotes oxidative stress and facilitates cell death, thereby contributing to the development and the progression of several mitochondria‐related diseases. Accordingly, compounds that reverse mitochondrial fragmentation could have therapeutic potential in treating such diseases. BGP‐15, a hydroxylamine derivative, prevents insulin resistance in humans and protects against several oxidative stress‐related diseases in animal models. Here we show that BGP‐15 promotes mitochondrial fusion by activating optic atrophy 1 (OPA1), a GTPase dynamin protein that assist fusion of the inner mitochondrial membranes. Suppression of Mfn1, Mfn2 or OPA1 prevents BGP‐15‐induced mitochondrial fusion. BGP‐15 activates Akt, S6K, mTOR, ERK1/2 and AS160, and reduces JNK phosphorylation which can contribute to its protective effects. Furthermore, BGP‐15 protects lung structure, activates mitochondrial fusion, and stabilizes cristae membranes in vivo determined by electron microscopy in a model of pulmonary arterial hypertension. These data provide the first evidence that a drug promoting mitochondrial fusion in in vitro and in vivo systems can reduce or prevent the progression of mitochondria‐related disorders.

Desethylamiodarone—A metabolite of amiodarone—Induces apoptosis on T24 human bladder cancer cells via multiple pathways

Bladder cancer (BC) is a common malignancy of the urinary tract that has a higher frequency in men than in women. Cytostatic resistance and metastasis formation are significant risk factors in BC therapy; therefore, there is great interest in overcoming drug resistance and in initiating research for novel chemotherapeutic approaches. Here, we suggest that desethylamiodarone (DEA)–a metabolite of amiodarone—may have cytostatic potential. DEA activates the collapse of mitochondrial membrane potential (detected by JC-1 fluorescence), and induces cell death in T24 human transitional-cell bladder carcinoma cell line at physiologically achievable concentrations. DEA induces cell cycle arrest in the G0/G1 phase, which may contribute to the inhibition of cell proliferation, and shifts the Bax/Bcl-2 ratio to initiate apoptosis, induce AIF nuclear translocation, and activate PARP-1 cleavage and caspase-3 activation. The major cytoprotective kinases—ERK and Akt—are inhibited by DEA, which may contribute to its cell death-inducing effects. DEA also inhibits the expression of B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1) and reduces colony formation of T24 bladder carcinoma cells, indicating its possible inhibitory effect on metastatic potential. These data show that DEA is a novel anti-cancer candidate of multiple cell death-inducing effects and metastatic potential. Our findings recommend further evaluation of its effects in clinical studies.

Evidence on Cholesterol-Controlled Lipid Raft Interaction of the Small Heat Shock Protein HSPB11

Small heat-shock proteins (sHSPs) are members of the family of molecular chaperones. Their major cellular function is considered to be the prevention of irreversible protein aggregation during stress conditions and subsequent promotion of the folding of partially denatured proteins. However, sHSPs may also be associated with biological membranes and participate in cellular “stress management” by acting as membrane-stabilizing factors. In spite of the great potential significance in the development of therapeutic strategies, the mechanisms of the membrane (and lipid) association of sHSPs are still unknown. A novel 16.2 kDa human sHSP, HSPB11, inhibits H2O2, taxol and etoposide-induced cell death through stabilization of the mitochondrial membrane system, the activation of HSP90, the stabilization of lipid rafts and activation of the PI-3-kinase—Akt cytoprotective pathway. We show here that HSPB11 binds to lipid membranes via a specific cholesterol-mediated interaction. The affinity of HSPB11 demonstrates a very distinct cholesterol-dependent binding to cholesterol/sphingomyelin Langmuir monolayers: If the cholesterol concentration increases above a certain level, HSPB11 binds to membranes much more efficiently. The possible roles of HSPB11 and other sHSPs in protection against stress-induced hydrophobic membrane defects are discussed.