FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2085644295> ?p ?o ?g. }

• W2085644295 endingPage "25710" @default.
• W2085644295 startingPage "25699" @default.
• W2085644295 abstract "Cell death by necrosis is emerging not merely as a passive phenomenon but as a cell-regulated process. Here, by using different necrotic triggers, we prove the existence of two distinct necrotic pathways. The mitochondrial reactive oxygen species generator 2,3-dimethoxy-1,4-naphthoquinone elicits necrosis characterized by the involvement of RIP1 and Drp1. However, G5, a non-selective isopeptidase inhibitor, triggers a distinct necrotic pathway that depends on the protein phosphatase PP2A and the actin cytoskeleton. PP2A catalytic subunit is stabilized by G5 treatment, and its activity is increased. Furthermore, PP2Ac accumulates into the cytoplasm during necrosis similarly to HMGB1. We have also defined in the actin-binding protein cofilin-1 a link between PP2A, actin cytoskeleton, and necrotic death. Cofilin-1-severing/depolymerization activity is negatively regulated by phosphorylation of serine 3. PP2A contributes to the dephosphorylation of serine 3 elicited by G5. Finally, a cofilin mutant that mimics phosphorylated Ser-3 can partially rescue necrosis in response to G5. Cell death by necrosis is emerging not merely as a passive phenomenon but as a cell-regulated process. Here, by using different necrotic triggers, we prove the existence of two distinct necrotic pathways. The mitochondrial reactive oxygen species generator 2,3-dimethoxy-1,4-naphthoquinone elicits necrosis characterized by the involvement of RIP1 and Drp1. However, G5, a non-selective isopeptidase inhibitor, triggers a distinct necrotic pathway that depends on the protein phosphatase PP2A and the actin cytoskeleton. PP2A catalytic subunit is stabilized by G5 treatment, and its activity is increased. Furthermore, PP2Ac accumulates into the cytoplasm during necrosis similarly to HMGB1. We have also defined in the actin-binding protein cofilin-1 a link between PP2A, actin cytoskeleton, and necrotic death. Cofilin-1-severing/depolymerization activity is negatively regulated by phosphorylation of serine 3. PP2A contributes to the dephosphorylation of serine 3 elicited by G5. Finally, a cofilin mutant that mimics phosphorylated Ser-3 can partially rescue necrosis in response to G5. Cell death by necrosis is characterized by organelle swelling and plasma membrane rupture, with the consequent release of cellular components in the microenvironment and the activation of the inflammatory response (1.Vanlangenakker N. Vanden Berghe T. Vandenabeele P. Many stimuli pull the necrotic trigger, an overview.Cell. Death Differ. 2012; 19: 75-86Crossref PubMed Scopus (304) Google Scholar). Until recently necrosis was considered a passive accidental type of cell death generally observed in the presence of severe cellular stress/damage. More recently, data have been accumulated pointing to the existence of a regulated necrotic response, with dedicated genes and signaling pathways (2.Vanden Berghe T. Linkermann A. Jouan-Lanhouet S. Walczak H. Vandenabeele P. Regulated necrosis: the expanding network of non-apoptotic cell death pathways.Nat. Rev. Mol. Cell Biol. 2014; 15: 135-147Crossref PubMed Scopus (1144) Google Scholar). TNF-α (tumor necrosis factor) has been helpful in discovering the cellular components controlling a regulated version of necrotic death. Under particular circumstances this cytokine can trigger necrosis (3.Laster S.M. Wood J.G. Gooding L.R. Tumor necrosis factor can induce both apoptic and necrotic forms of cell lysis.J. Immunol. 1988; 141: 2629-2634Crossref PubMed Google Scholar). TNF-α can stimulate the assembling of the necrosome, a signaling complex that includes the kinases RIP1, 3The abbreviations used are: RIP1receptor-interacting protein 1MLKLmixed lineage kinase-likePP2Aprotein phosphatase 2ANec-1necrostatin-1DMNQ2,3-dimethoxy-1,4-naphthoquinoneDiFMUP6,8-difluoro-4-methylumbelliferyl phosphateTRITCtetramethylrhodamine isothiocyanateNSAnecrosulfonamideBoc-fmkbutoxycarbonyl-fluoromethyl ketoneSmacsecond mitochondria-derived activator of caspases. R1P3, and MLKL (mixed lineage kinase domain-like) (4.Sun L. Wang H. Wang Z. He S. Chen S. Liao D. Wang L. Yan J. Liu W. Lei X. Wang X. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase.Cell. 2012; 148: 213-227Abstract Full Text Full Text PDF PubMed Scopus (1664) Google Scholar, 5.Cho Y.S. Challa S. Moquin D. Genga R. Ray T.D. Guildford M. Chan F.K. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation.Cell. 2009; 137: 1112-1123Abstract Full Text Full Text PDF PubMed Scopus (1687) Google Scholar, 6.Li J. McQuade T. Siemer A.B. Napetschnig J. Moriwaki K. Hsiao Y.S. Damko E. Moquin D. Walz T. McDermott A. Chan F.K. Wu H. The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis.Cell. 2012; 150: 339-350Abstract Full Text Full Text PDF PubMed Scopus (779) Google Scholar, 7.Cai Z. Jitkaew S. Zhao J. Chiang H.C. Choksi S. Liu J. Ward Y. Wu L.G. Liu Z.G. Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis.Nat. Cell Biol. 2014; 16: 55-65Crossref PubMed Scopus (762) Google Scholar). This complex is regulated by phosphorylation, acetylation, and ubiquitination (5.Cho Y.S. Challa S. Moquin D. Genga R. Ray T.D. Guildford M. Chan F.K. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation.Cell. 2009; 137: 1112-1123Abstract Full Text Full Text PDF PubMed Scopus (1687) Google Scholar, 8.Moriwaki K. Chan F.K. RIP3: a molecular switch for necrosis and inflammation.Genes Dev. 2013; 27: 1640-1649Crossref PubMed Scopus (244) Google Scholar). Necrosome-elicited death response has been defined as necroptosis, and the RIP1 inhibitor necrostatin has been instrumental in unveiling the contribution of necroptosis under different death circumstances (9.Degterev A. Hitomi J. Germscheid M. Ch'en I.L. Korkina O. Teng X. Abbott D. Cuny G.D. Yuan C. Wagner G. Hedrick S.M. Gerber S.A. Lugovskoy A. Yuan J. Identification of RIP1 kinase as a specific cellular target of necrostatins.Nat. Chem. Biol. 2008; 4: 313-321Crossref PubMed Scopus (1466) Google Scholar). receptor-interacting protein 1 mixed lineage kinase-like protein phosphatase 2A necrostatin-1 2,3-dimethoxy-1,4-naphthoquinone 6,8-difluoro-4-methylumbelliferyl phosphate tetramethylrhodamine isothiocyanate necrosulfonamide butoxycarbonyl-fluoromethyl ketone second mitochondria-derived activator of caspases. Mitochondrial dysfunctions also mark necrosis. It was proposed that the necrosome could influence mitochondrial shape and function through the modulation of Drp1, a GTPase that controls mitochondrial fission. The mitochondrial protein PGAM5 (phosphoglycerate mutase 5), which is under the control of MLKL, could act as a downstream effector of the necroptotic signaling by dephosphorylating and thus activating Drp1 (10.Wang Z. Jiang H. Chen S. Du F. Wang X. The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways.Cell. 2012; 148: 228-243Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar). This model is debated, and recently the contribution of Drp1 to necroptosis has been questioned (11.Moujalled D.M. Cook W.D. Murphy J.M. Vaux D.L. Necroptosis induced by RIPK3 requires MLKL but not Drp1.Cell Death Dis. 2014; 5: e1086Crossref PubMed Scopus (73) Google Scholar, 12.Remijsen Q. Goossens V. Grootjans S. Van den Haute C. Vanlangenakker N. Dondelinger Y. Roelandt R. Bruggeman I. Goncalves A. Bertrand M.J. Baekelandt V. Takahashi N. Berghe T.V. Vandenabeele P. Depletion of RIPK3 or MLKL blocks TNF-driven necroptosis and switches towards a delayed RIPK1 kinase-dependent apoptosis.Cell Death Dis. 2014; 5: e1004Crossref PubMed Scopus (144) Google Scholar). Although some hints have accumulated on cytokine- and pathogen-induced necrosis, other varieties, engaged by cellular stresses, triggered by physical agents or chemicals are still obscure (2.Vanden Berghe T. Linkermann A. Jouan-Lanhouet S. Walczak H. Vandenabeele P. Regulated necrosis: the expanding network of non-apoptotic cell death pathways.Nat. Rev. Mol. Cell Biol. 2014; 15: 135-147Crossref PubMed Scopus (1144) Google Scholar). In addition to elements of the necroptotic pathway, further players could be involved in the stress-induced necrotic response. Lysosomal proteases released into the cytosol after lysosomal membrane permeabilization (13.Boya P. Kroemer G. Lysosomal membrane permeabilization in cell death.Oncogene. 2008; 27: 6434-6451Crossref PubMed Scopus (1039) Google Scholar) and calpains, proteolytic enzymes activated by increase of Ca2+, have been proposed as necrotic regulators (14.Arnandis T. Ferrer-Vicens I. García-Trevijano E.R. Miralles V.J. García C. Torres L. Viña J.R. Zaragozá R. Calpains mediate epithelial-cell death during mammary gland involution: mitochondria and lysosomal destabilization.Cell Death Differ. 2012; 19: 1536-1548Crossref PubMed Scopus (44) Google Scholar, 15.Moubarak R.S. Yuste V.J. Artus C. Bouharrour A. Greer P.A. Menissier-de Murcia J. Susin S.A. Sequential activation of poly(ADP-ribose) polymerase 1, calpains, and Bax is essential in apoptosis-inducing factor-mediated programmed necrosis.Mol. Cell. Biol. 2007; 27: 4844-4862Crossref PubMed Scopus (276) Google Scholar). In particular, the poly(ADP-ribose) polymerase 1 (PARP-1) and cyclophilin D (CypD) have been linked in some studies to RIP1 in models of oxidative stress-induced necrosis (16.Xu Y. Huang S. Liu Z.G. Han J. Poly(ADP-ribose) polymerase-1 signaling to mitochondria in necrotic cell death requires RIP1/TRAF2-mediated JNK1 activation.J. Biol. Chem. 2006; 281: 8788-8795Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 17.Temkin V. Huang Q. Liu H. Osada H. Pope R.M. Inhibition of ADP/ATP exchange in receptor-interacting protein-mediated necrosis.Mol. Cell. Biol. 2006; 26: 2215-2225Crossref PubMed Scopus (166) Google Scholar). It has been proposed that the phosphoglycerate mutase 5 (PGAM5)/Drp1 axis could represent a convergent node for different necrotic pathways (10.Wang Z. Jiang H. Chen S. Du F. Wang X. The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways.Cell. 2012; 148: 228-243Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar). To clarify whether or not multiple necrotic pathways exist, we have compared the necrotic responses elicited by two different chemical stresses. Our studies indicate the existence of different necrotic pathways and identify in the protein phosphatase 2A (PP2A)-cofilin-actin axis a new regulator of a specific form of necrotic death. U87MG, U-118MG, HT29, and IMR90-E1A/Bcl2/C9DN cells were grown in DMEM supplemented with 10% FBS, penicillin (100 units/ml), glutamine (2 mmol/liter), and streptomycin (100 μg/ml) at 37 °C in 5% CO2 atmosphere. U87MG cells expressing Bcl-xL, Drp1 K38A, GFP, Cofilin constructs, and myristoylated Akt were generated by retroviral infection (18.Foti C. Florean C. Pezzutto A. Roncaglia P. Tomasella A. Gustincich S. Brancolini C. Characterization of caspase-dependent and caspase-independent deaths in glioblastoma cells treated with inhibitors of the ubiquitin-proteasome system.Mol. Cancer Ther. 2009; 8: 3140-3150Crossref PubMed Scopus (20) Google Scholar). In all trypan blue exclusion assays, at least 400 cells from three independent samples were counted. RNA oligos for interference (RNAi) were purchased from Invitrogen, Dharmacon, and Qiagen. Cells were transfected 24 h after plating by adding the Opti-MEM medium containing Lipofectamine 2000 (Invitrogen) plus RNAi oligos. The following chemicals were used: LY (LY294002; LC Laboratories), BOC-D(OMe)-fmk (Imgenex), bortezomib (LC Laboratories), necrostatin-1 (Nec-1) (Enzo Life Sciences), cytochalasin D, cucurbitacin E, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), Mdivi-1, biotinylated concanavalin A, and DMSO (Sigma), MitoTracker Red/CMXRos, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), streptavidin-FITC, phalloidin-TRITC (Invitrogen/Molecular Probes), okadaic acid (Enzo Life Sciences), and necrosulfonamide (NSA) (Merck Millipore). Primary antibodies were anti-FLAG and anti-actin (Sigma), anti-nucleoporin p62, anti-Ran (RAS-related nuclear protein), anti-Bcl-xL, anti-Drp1 (DLP1) (BD Biosciences), anti-JNK Thr(P)-183/Tyr-185, anti-JNK, anti-p38, anti-pp38, anti-Erk, anti-pErk, anti-Akt, anti-Akt p473, anti-Akt p308 (Cell Signaling), anti-F1-ATP synthase, anti-cofilin-1, anti-pCofilin-1 (Santa Cruz Biotechnologies), anti-Smac/DIABLO (19.Henderson C.J. Aleo E. Fontanini A. Maestro R. Paroni G. Brancolini C. Caspase activation and apoptosis in response to proteasome inhibitors.Cell Death Differ. 2005; 12: 1240-1254Crossref PubMed Scopus (45) Google Scholar), anti-PP2A/C subunit, anti-PP2A/A subunit (Upstate Biotechnology, Lake Placid, NY), and anti-HMGB1 (AbCam, Cambridge, UK). Secondary anti-mouse and anti-rabbit antibodies were Alexa Fluor 488- and Alexa Fluor 546-conjugated (Invitrogen). Cell lysates after SDS/PAGE were transferred to a 0.2-μm nitrocellulose membrane and incubated with the specific primary antibodies. For primary antibody stripping, blots were incubated for 30 min at 60 °C in stripping solution (62.5 mm Tris-HCl, pH 6.8, 2% SDS, 100 mm β-mercaptoethanol). Quantitative densitometric analysis of the immunoblots was performed by ChemiDoc software (Bio-Rad). Cells were lysed directly in Petri dishes with lysis buffer (50 mm Tris-HCl, pH 8.0, 100 mm NaCl, 2 mm EDTA, and 1% (v/v) Igepal CA-630, PMSF, and a protease inhibitor mixture (Sigma). Whole cell lysates were incubated with anti-PP2Ac antibody (ON). After incubation with protein A beads the immunocomplexes were washed four times with lysis buffer. Beads were resuspended in phosphatase assay buffer (50 mm Tris-HCl, pH 7.0, and 0.1 mm CaCl2), incubated for 30 min at 37 °C with or without okadaic acid 100 nm in presence of 1 mm NiCl2 and 0.125 mg/ml BSA, and then analyzed by fluorimetric assay using DiFMUP substrate as described (20.Wegner A.M. McConnell J.L. Blakely R.D. Wadzinski B.E. An automated fluorescence-based method for continuous assay of PP2A activity.Methods Mol. Biol. 2007; 365: 61-69PubMed Google Scholar). Immunocomplexes were recollected after the fluorimetric assay and next resolved in SDS/PAGE electrophoresis and immunoblotted. Mitochondria were labeled in vivo for 1 h with 25 nmol/liter MitoTracker Red. Cells were fixed with 3% paraformaldehyde and permeabilized with 0.5% Triton X-100 and incubated with the primary antibody, phalloidin-TRITC. After washes, coverslips were incubated with the relative secondary antibodies. Cells were imaged with a Leica confocal scanner SP equipped with a 488 λ Ar laser and a 543–633 λ HeNe laser. The image analysis was performed using the MetaMorph 6.04 software. Cell images for deconvolution were taken using the Leica AF6000 LX microscope. Deconvolution software was used for image deconvolution and three-dimensional view reconstruction. Cells were lysed using TRI-REAGENT (Molecular Research Center). 1.0 μg of total RNA was retro-transcribed by using 100 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen). Quantitative real-time-PCRs were performed using the Bio-Rad CFX96 and SYBR Green technology. Data were analyzed by a comparative threshold cycle using hypoxanthine-guanine phosphoribosyltransferase and β-actin as normalizer genes. All reactions were done in triplicate. Results are expressed as the means ± S.D. Student's t test was performed with Excel software. p values are represented as: *, p < 0.05; **, p < 0.01; ***, p < 0.005. Data of the spreading area were analyzed using Non-parametric Mann-Whitney test (Prism GraphPad Software); ***, p < 0.0001. To explore the existence of different necrotic signaling pathways, we used two different chemical stressors: the isopeptidases inhibitor G5, an inducer of alterations in cell adhesion and actin cytoskeleton (18.Foti C. Florean C. Pezzutto A. Roncaglia P. Tomasella A. Gustincich S. Brancolini C. Characterization of caspase-dependent and caspase-independent deaths in glioblastoma cells treated with inhibitors of the ubiquitin-proteasome system.Mol. Cancer Ther. 2009; 8: 3140-3150Crossref PubMed Scopus (20) Google Scholar, 21.Aleo E. Henderson C.J. Fontanini A. Solazzo B. Brancolini C. Identification of new compounds that trigger apoptosome-independent caspase activation and apoptosis.Cancer Res. 2006; 66: 9235-9244Crossref PubMed Scopus (81) Google Scholar, 22.Fontanini A. Foti C. Potu H. Crivellato E. Maestro R. Bernardi P. Demarchi F. Brancolini C. The isopeptidase inhibitor G5 triggers a caspase-independent necrotic death in cells resistant to apoptosis: a comparative study with the proteasome inhibitor bortezomib.J. Biol. Chem. 2009; 284: 8369-8381Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), and DMNQ, a generator of reactive oxygen species at mitochondrial level (23.Tchivilev I. Madamanchi N.R. Vendrov A.E. Niu X.L. Runge M.S. Identification of a protective role for protein phosphatase 1cγ1 against oxidative stress-induced vascular smooth muscle cell apoptosis.J. Biol. Chem. 2008; 283: 22193-22205Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). As the cellular model to study necrosis we selected U87MG glioblastoma cells because of their intrinsic resistance to apoptosis and the tendency to die by necrosis (18.Foti C. Florean C. Pezzutto A. Roncaglia P. Tomasella A. Gustincich S. Brancolini C. Characterization of caspase-dependent and caspase-independent deaths in glioblastoma cells treated with inhibitors of the ubiquitin-proteasome system.Mol. Cancer Ther. 2009; 8: 3140-3150Crossref PubMed Scopus (20) Google Scholar). In addition, we overexpressed Bcl-xL to further suppress apoptosis. Cells were treated with escalating doses of G5 or DMNQ, and cell death was scored by a trypan blue assay (Fig. 1A). In general 5 μm G5 and 20 μm DMNQ were employed for the subsequent studies. Contrary to bortezomib, G5- or DMNQ-induced cell death was unaffected by caspase inhibitors (Fig. 1B). Next we evaluated the mitochondrial morphology using MitoTracker in relation to mitochondrial outer membrane permeabilization. As a marker of mitochondrial outer membrane permeabilization, we explored Smac localization. Both necrotic stimuli induced a dramatic mitochondrial fragmentation (Fig. 1C), with the quantitative analysis reported in Fig. 1D. Smac was usually retained in the mitochondria, thus proving the absence of mitochondrial outer membrane permeabilization (Fig. 1E). Curiously in the case of DMNQ a fraction of cells (10%) presented intact mitochondrial outer membrane but decreased MitoTracker staining, thus indicating that Δψm collapse occurs before mitochondrial outer membrane permeabilization (Fig. 1F). Cytoplasmic vacuolization, as a consequence of endoplasmic reticulum stress, occurs in response to the engagement of the unfolded response by UPS inhibitors (24.Mimnaugh E.G. Xu W. Vos M. Yuan X. Isaacs J.S. Bisht K.S. Gius D. Neckers L. Simultaneous inhibition of hsp 90 and the proteasome promotes protein ubiquitination, causes endoplasmic reticulum-derived cytosolic vacuolization, and enhances antitumor activity.Mol. Cancer Ther. 2004; 3: 551-566Crossref PubMed Scopus (29) Google Scholar). As shown in Fig. 1G, glioma cells incubated with G5 exhibit cytoplasmic vacuolization. However, when a time course analysis was performed, it was evident that mitochondrial fragmentation precedes cytoplasmic vacuolization (Fig. 1H). In conclusion, mitochondrial fragmentation is an early event during cell death induced either by G5 or DMNQ treatment. To elucidate the contribution of mitochondrial fragmentation to G5- and DMNQ-induced necrosis, cells were treated with the Drp1 inhibitor Mdivi-1 (25.Cassidy-Stone A. Chipuk J.E. Ingerman E. Song C. Yoo C. Kuwana T. Kurth M.J. Shaw J.T. Hinshaw J.E. Green D.R. Nunnari J. Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization.Dev. Cell. 2008; 14: 193-204Abstract Full Text Full Text PDF PubMed Scopus (817) Google Scholar). Mdivi-1 attenuated necrosis only in response to DMNQ (Fig. 2A). To confirm this result we generated U87MG/Bcl-xL cells expressing a dominant negative mutant (K38A) of Drp1 (Fig. 2B). Treatment with G5 or with DMNQ of Drp1-K38A-overexpressing cells confirmed the result obtained with the Drp1 inhibitor. The dependence from Drp1 activity to fully elicit necrosis was observed only in the case of DMNQ treatment (Fig. 2C). To further prove that necrosis elicited by DMNQ or G5 engages two distinct pathways, we evaluated the contribution of RIP1 by treating cells with the specific inhibitor Nec-1 (9.Degterev A. Hitomi J. Germscheid M. Ch'en I.L. Korkina O. Teng X. Abbott D. Cuny G.D. Yuan C. Wagner G. Hedrick S.M. Gerber S.A. Lugovskoy A. Yuan J. Identification of RIP1 kinase as a specific cellular target of necrostatins.Nat. Chem. Biol. 2008; 4: 313-321Crossref PubMed Scopus (1466) Google Scholar). Cell death was efficiently rescued by Nec-1 only when elicited by DMNQ (Fig. 2D). These results demonstrate that two distinct necrotic pathways are activated by DMNQ and G5. We also investigated the ability of NSA, a MLKL inhibitor that blocks necrosis downstream of RIP3 activation (4.Sun L. Wang H. Wang Z. He S. Chen S. Liao D. Wang L. Yan J. Liu W. Lei X. Wang X. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase.Cell. 2012; 148: 213-227Abstract Full Text Full Text PDF PubMed Scopus (1664) Google Scholar), to differentially influence G5- and DMNQ-induced necrosis. Inhibition of MLKL did not suppress the appearance of cell death in response to G5 and, surprisingly, in DMNQ-treated cells as well (Fig. 2E). To prove inhibitor efficiency, we treated U87MG/Bcl-xL cells with a classic necroptotic stimulus: the combination TNF-α, Smac mimetic (26.Lecis D. Mastrangelo E. Belvisi L. Bolognesi M. Civera M. Cossu F. De Cesare M. Delia D. Drago C. Manenti G. Manzoni L. Milani M. Moroni E. Perego P. Potenza D. Rizzo V. Scavullo C. Scolastico C. Servida F. Vasile F. Seneci P. Dimeric Smac mimetics/IAP inhibitors as in vivo active pro-apoptotic agents. Part II: structural and biological characterization.Bioorg. Med. Chem. 2012; 20: 6709-6723Crossref PubMed Scopus (26) Google Scholar), and Boc-fmk, as caspase inhibitor). U87MG cells were unresponsive to the TNF-α, Smac mimetic, and Boc-fmk combination (Fig. 2F). We also used cyclohexamide instead of Smac in the combination (TNF-α, cyclohexamide, Boc-fmk). Here again cell death was undetectable (Fig. 2G). Finally we used staurosporine, a recently suggested necroptotic trigger (27.Dunai Z.A. Imre G. Barna G. Korcsmaros T. Petak I. Bauer P.I. Mihalik R. Staurosporine induces necroptotic cell death under caspase-compromised conditions in U937 cells.PLoS ONE. 2012; 7: e41945Crossref PubMed Scopus (85) Google Scholar). Staurosporine induced both apoptosis and necrosis in U87MG/Bcl-xL cells (Fig. 2H); however, this necrotic response was unaffected by RIP1 and MLKL inhibition. When the same necrotic triggers were evaluated in IMR90-E1A cells, the vast majority of the cells died by apoptosis, as proved by the effectiveness of the caspase inhibitor (Fig. 2, I–K). We also examined colon cancer HT29 cells, commonly utilized for necroptotic experiments. These cells died by necroptosis in response to the TNF-α, Smac mimetic, and Boc-fmk combination. Nec-1 efficiently abrogated this cell death, and NSA was active, although less potent compared with Nec-1 (Fig. 2L). These results prove that HT29 cells can die through necroptosis and that the two inhibitors are valuable. When we analyzed G5- and DMNQ-induced death in HT29 cells, similarly to U87MG cells the two drugs exhibited differential susceptibility to RIP1 and MLKL inhibition. G5-induced cell death was unaffected, whereas DMNQ was partially counteracted by the two inhibitors, with Nec-1 showing a stronger effect (Fig. 2M). Overall these results further confirm the existence of different necrotic responses. Finally, we discovered that the divergent responsiveness of HT29 and U87MG cells to necroptotic stimuli and the different responsiveness to MLKL inhibition, in the case of DMNQ-induced necrosis, could be explained by the differential expression of RIP3. In fact, when we interrogated public available gene expression profiles from U87MG and HT29 cells for necroptotic gene mRNA levels (Fig. 2N), RIP3 mRNA was clearly reduced in U87MG compared with HT29 cells. Quantitative real-time-PCR experiments proved the dramatic reduction (∼400 times respect to HT29 cells) of RIP3 mRNA in U87MG cells (Fig. 2O). To gain insight into the necrotic pathway elicited by G5, we decided to monitor the activation of signaling pathways transducing stress and prosurvival signals (28.Kyriakis J.M. Avruch J. Mammalian MAPK signal transduction pathways activated by stress and inflammation: a 10-year update.Physiol. Rev. 2012; 92: 689-737Crossref PubMed Scopus (949) Google Scholar) in G5-treated cells. We evaluated activations of ERKs, p38, JUNK, and Akt. ERKs and p38 but not JUNK were transiently activated in response to G5. However, these activations were anticipated without mirroring the appearance of necrosis (Fig. 3A). When the status of Akt activation was evaluated, it emerged that the kinetic of dephosphorylation of serine 473 and also of threonine 308 (after a transient up-regulation) was paired to the appearance of necrosis (Fig. 2E). This result suggests that inhibition of Akt could be linked to G5-induced necrosis. PP2A can dephosphorylate and inactivate Akt (29.Kuo Y.C. Huang K.Y. Yang C.H. Yang Y.S. Lee W.Y. Chiang C.W. Regulation of phosphorylation of Thr-308 of Akt, cell proliferation, and survival by the B55α regulatory subunit targeting of the protein phosphatase 2A holoenzyme to Akt.J. Biol. Chem. 2008; 283: 1882-1892Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 30.Li G. Ji X.D. Gao H. Zhao J.S. Xu J.F. Sun Z.J. Deng Y.Z. Shi S. Feng Y.X. Zhu Y.Q. Wang T. Li J.J. Xie D. EphB3 suppresses non-small-cell lung cancer metastasis via a PP2A/RACK1/Akt signalling complex.Nat. Commun. 2012; 3: 667Crossref PubMed Scopus (90) Google Scholar). Hence, we monitored PP2A subunit levels in cells treated with G5. Levels of PP2Ac catalytic subunit were increased until 3 h from G5 treatment with a drop at 6 h, after the advent of necrosis. By contrast levels of the scaffold subunit A (PR65) were unaffected. Having discovered an increase of the PP2Ac subunit, we next evaluated whether PP2A enzymatic activity was augmented after G5 treatment, thus explaining Akt dephosphorylation and inactivation. Fig. 3B illustrates that PP2A phosphatase activity is augmented in cells treated with G5. To evaluate the contribution of PP2A to G5-induced necrosis, we silenced the expression of the catalytic subunit and next incubated U87MG/Bcl-xL cells with G5 or DMNQ. Down-regulation of PP2Ac impacted both necrotic responses, although with opposite effects (Fig. 4A). PP2Ac is required for necrosis in response to G5, but it counteracts necrosis in response to DMNQ. This evidence further strengthens the hypothesis that cells can engage multiple necrotic pathways. The impact of PP2A in G5-induced necrosis was also evident at the level of mitochondrial fragmentation (Fig. 4B). In cells with reduced PP2Ac expression, mitochondria fragmentation is much less pronounced. To confirm the role of PP2A we used another siRNA that targets a different region of PP2Ac. Again, down-regulation of PP2Ac limited necrosis in response to G5 and favored necrosis in response to DMNQ (Fig. 4C). The impact of PP2A on G5-induced necrosis could be related to the status of Akt activation; hence, we evaluated Akt phosphorylation in cells silenced for PP2Ac and treated with G5 for 1 h. Fig. 4D and the quantitative analysis in Fig. 4E evidence that phosphorylation at threonine 308 was higher after G5 treatment in PP2Ac-silenced cells. In addition to its well established anti-apoptotic role, Akt has been reported to counteract some kinds of necrotic death (31.Mochizuki T. Asai A. Saito N. Tanaka S. Katagiri H. Asano T. Nakane M. Tamura A. Kuchino Y. Kitanaka C. Kirino T. Akt protein kinase inhibits non-apoptotic programmed cell death induced by ceramide.J. Biol. Chem. 2002; 277: 2790-2797Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 32.Liu Q. Qiu J. Liang M. Golinski J. van Leyen K. Jung J.E. You Z. Lo E.H. Degterev A. Whalen M.J. Akt and mTOR mediate programmed necrosis in neurons.Cell Death Dis. 2014; 5: e1084Crossref PubMed Scopus (107) Google Scholar). Hence, we explored whether the pro-necrotic role of PP2A in G5-treated cells could be explicated through the inhibition of Akt activity. First, by using the PI3K inhibitor LY, we observed that suppression of the PI3K-Akt axis was insufficient for triggering the death of U87MG/Bcl-xL cells (Fig. 4, F and G). Second, G5-induced necrosis was unaffected by the overexpression of a constitutive active form of Akt (Fig. 4, H and I). In summary, although Akt activity is down-regulated by G5 treatment and PP2A contributes to this modulation, this kinase does not play a major role during G5-induced necrotic death. The contribution of PP2A to G5-induced necrosis was confirmed in U-118MG cells, another glioblastoma cell line (Fig. 4J). By contrast, in a different apoptosis-resistant cell line represented by human fibroblasts expressing E1A and Bcl2 oncogenes and a catalytic inactive dominant negative mutant of Caspase-9 (19.Henderson C.J. Aleo E. Fontanini A. Maestro R. Paroni G. Brancolini C. Caspase activation and apoptosis in response to proteasome inhibitors.Cell" @default.
• W2085644295 created "2016-06-24" @default.
• W2085644295 creator A5033589027 @default.
• W2085644295 creator A5051699449 @default.
• W2085644295 creator A5080222062 @default.
• W2085644295 date "2014-09-01" @default.
• W2085644295 modified "2023-10-14" @default.
• W2085644295 title "A Receptor-interacting Protein 1 (RIP1)-independent Necrotic Death under the Control of Protein Phosphatase PP2A That Involves the Reorganization of Actin Cytoskeleton and the Action of Cofilin-1" @default.
• W2085644295 cites W1966880102 @default.
• W2085644295 cites W1970560403 @default.
• W2085644295 cites W1973063454 @default.
• W2085644295 cites W1975087901 @default.
• W2085644295 cites W1976312989 @default.
• W2085644295 cites W1978340143 @default.
• W2085644295 cites W1979032223 @default.
• W2085644295 cites W1987213099 @default.
• W2085644295 cites W1988860478 @default.
• W2085644295 cites W1992060900 @default.
• W2085644295 cites W1997012817 @default.
• W2085644295 cites W1998983498 @default.
• W2085644295 cites W2003736664 @default.
• W2085644295 cites W2004266769 @default.
• W2085644295 cites W2005749687 @default.
• W2085644295 cites W2009277812 @default.
• W2085644295 cites W2012516837 @default.
• W2085644295 cites W2012859385 @default.
• W2085644295 cites W2022894667 @default.
• W2085644295 cites W2031853870 @default.
• W2085644295 cites W2041580366 @default.
• W2085644295 cites W2045852899 @default.
• W2085644295 cites W2052950446 @default.
• W2085644295 cites W2055453372 @default.
• W2085644295 cites W2056579482 @default.
• W2085644295 cites W2058436322 @default.
• W2085644295 cites W2059009119 @default.
• W2085644295 cites W2059833049 @default.
• W2085644295 cites W2061496561 @default.
• W2085644295 cites W2072585359 @default.
• W2085644295 cites W2078463367 @default.
• W2085644295 cites W2080878773 @default.
• W2085644295 cites W2081053802 @default.
• W2085644295 cites W2082870439 @default.
• W2085644295 cites W2082919112 @default.
• W2085644295 cites W2091363184 @default.
• W2085644295 cites W2104028914 @default.
• W2085644295 cites W2113944758 @default.
• W2085644295 cites W2120723414 @default.
• W2085644295 cites W2121000910 @default.
• W2085644295 cites W2123504406 @default.
• W2085644295 cites W2132161879 @default.
• W2085644295 cites W2133169035 @default.
• W2085644295 cites W2135072453 @default.
• W2085644295 cites W2150446853 @default.
• W2085644295 cites W2151881294 @default.
• W2085644295 cites W2158662692 @default.
• W2085644295 cites W2159190959 @default.
• W2085644295 cites W4240253200 @default.
• W2085644295 doi "https://doi.org/10.1074/jbc.m114.575134" @default.
• W2085644295 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4162173" @default.
• W2085644295 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/25096578" @default.
• W2085644295 hasPublicationYear "2014" @default.
• W2085644295 type Work @default.
• W2085644295 sameAs 2085644295 @default.
• W2085644295 citedByCount "19" @default.
• W2085644295 countsByYear W20856442952015 @default.
• W2085644295 countsByYear W20856442952016 @default.
• W2085644295 countsByYear W20856442952017 @default.
• W2085644295 countsByYear W20856442952018 @default.
• W2085644295 countsByYear W20856442952020 @default.
• W2085644295 countsByYear W20856442952021 @default.
• W2085644295 countsByYear W20856442952022 @default.
• W2085644295 countsByYear W20856442952023 @default.
• W2085644295 crossrefType "journal-article" @default.
• W2085644295 hasAuthorship W2085644295A5033589027 @default.
• W2085644295 hasAuthorship W2085644295A5051699449 @default.
• W2085644295 hasAuthorship W2085644295A5080222062 @default.
• W2085644295 hasBestOaLocation W20856442951 @default.
• W2085644295 hasConcept C11960822 @default.
• W2085644295 hasConcept C125705527 @default.
• W2085644295 hasConcept C142669718 @default.
• W2085644295 hasConcept C1491633281 @default.
• W2085644295 hasConcept C178666793 @default.
• W2085644295 hasConcept C185592680 @default.
• W2085644295 hasConcept C2776601116 @default.
• W2085644295 hasConcept C2993400109 @default.
• W2085644295 hasConcept C389152 @default.
• W2085644295 hasConcept C55493867 @default.
• W2085644295 hasConcept C86803240 @default.
• W2085644295 hasConcept C95444343 @default.
• W2085644295 hasConceptScore W2085644295C11960822 @default.
• W2085644295 hasConceptScore W2085644295C125705527 @default.
• W2085644295 hasConceptScore W2085644295C142669718 @default.
• W2085644295 hasConceptScore W2085644295C1491633281 @default.
• W2085644295 hasConceptScore W2085644295C178666793 @default.
• W2085644295 hasConceptScore W2085644295C185592680 @default.
• W2085644295 hasConceptScore W2085644295C2776601116 @default.
• W2085644295 hasConceptScore W2085644295C2993400109 @default.
• W2085644295 hasConceptScore W2085644295C389152 @default.

• next