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Landfill soil leachates, containing myriad of xenobiotics, increase genotoxic and cytotoxic stress-induced cell death. However, the underlying mechanism involved in the elimination of the damaged cells is yet to be fully elucidated. This study investigated the apoptotic processes induced in lymphoma (Jurkat) cells by landfill soil leachates from Olusosun (OSL, Nigeria) and Nagpur (NPL, India). Jurkat was incubated with sub-lethal concentrations of OSL and NPL for 24 h and analyzed for DNA fragmentation and apoptosis using agarose gel electrophoresis and Hoechst 33258-PI staining, respectively. Complementary DNA expression profiling of some pro-apoptotic and anti-apoptotic genes regulating apoptosis was also analyzed using real-time PCR (RT-PCR) method. Agarose gel electrophoresis revealed DNA fragmentations in OSL and NPL-treated cells. Hoecsht-33258 - Propidium Iodide (PI) based apoptotic analysis confirmed apoptotic cell death in exposed Jurkat. RT-PCR analysis revealed different fold changes in the pro- and anti-apoptotic genes in OSL and NPL-treated Jurkat. There was significant increase in fold change of the up-regulated genes; apoptosis inducing factor mitochondrion-associated 2 (AIFM2), Fas-associated death domain (FADD), Caspase-2, Caspase-6, BH3 interacting domain death agonist (BID), tumor suppressor (p53), and BCL2 associated agonist of cell death (BAD) and down-regulation of apoptosis inhibitor 5 (API5). Results suggest that OSL and NPL elicited genotoxic stress-related apoptosis in Jurkat. The dysregulation in the expression of genes involved in apoptotic processes in wildlife and human exposed to landfill emissions may increase aetiology of various pathological diseases including cancer.

The tumour‐suppressive role of LINC00472 has been extensively reported in various human cancers such as lung, colon and ovarian cancers, yet its function in pancreatic cancer remains unidentified. Here, the current research aimed to explore the role and regulatory axis mediated by LINC00472 in the progression of pancreatic cancer. RT‐qPCR was adopted to determine LINC00472 expression in the harvested pancreatic cancer tissues and adjacent normal tissues. Loss‐of‐function and gain‐of‐function experiments were performed to examine the effects of LINC00472 on proliferation and apoptosis in vitro and tumorigenesis in vivo. Immunoblotting was performed to detect the expression of several proliferation and apoptosis‐related proteins. Bioinformatic analysis, dual‐luciferase reporter assay and RNA pull‐down were conducted to profile the relationships between LINC00472 and miR‐23a‐3p, between miR‐23a‐3p and FOXO3 and between FOXO3 and BID. The LINC00472 expression was down‐regulated by ZEB1 in the pancreatic cancer cells and tissues. LINC00472 could competitively bind to miR‐23a‐3p to enhance the expression of FOXO3, which consequently could promote the BID expression, thereby suppressing proliferation and promoting the apoptosis of pancreatic cancer cells. Meanwhile, the inhibitory role of LINC00472 in tumorigenesis was validated in vivo, and the LINC00472‐mediated miR‐23a‐3p/FOXO3/BID axis was also demonstrated in the nude mouse tumour formation model. The study substantiated the antitumour activity of LINC00472 in pancreatic cancer and proposed a regulatory axis in which LINC00472 competitively binds to miR‐23a‐3p to enhance the FOXO3 expression and promote BID expression. Consequently, these findings provide theoretical basis for developing potential targets for the treatment of pancreatic cancer.

Alzheimer's disease (AD) is a neurodegenerative disorder disease, disturbing people's normal life. Syringin was mentioned to antagonize Amyloid-β (Aβ)-induced neurotoxicity. However, the action mechanism is still not fully elucidated. This study aimed to explore a molecular mechanism of syringin in defending Aβ-induced neurotoxicity. SK-N-SH and SK-N-BE cells were treated with amyloid β-protein fragment 25-35 (Aβ25-35) to induce cell neurotoxicity. The injury effects were distinguished by assessing cell viability and cell apoptosis using cell counting kit-8 (CCK-8) assay and flow cytometry assay, respectively. The expression of Cleaved-caspase3 (Cleaved-casp3), B cell lymphoma/leukemia-2 (Bcl-2), Bcl-2 associated X protein (Bax) and BH3 interacting domain death agonist (BID) at the protein level was determined by western blot. The expression of miR-124-3p and BID was detected using quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between miR-124-3p and BID was predicted by the online database starBase and confirmed by dual-luciferase reporter assay plus RNA pull-down assay. Aβ25-35 treatment inhibited cell viability and induced cell apoptosis, while the addition of syringin recovered cell viability and suppressed cell apoptosis. MiR-124-3p was significantly downregulated in Aβ25-35-treated SK-N-SH and SK-N-BE cells, and BID was upregulated. Nevertheless, the addition of syringin reversed their expression. BID was a target of miR-124-3p, and its downregulation partly prevented Aβ25-35-induced injuries. Syringin protected against Aβ25-35-induced neurotoxicity by enhancing miR-124-3p expression and weakening BID expression, and syringin strengthened the expression of miR-124-3p to diminish BID level. Syringin ameliorated Aβ25-35-induced neurotoxicity in SK-N-SH and SK-N-BE cells by regulating miR-124-3p/BID pathway, which could be a novel theoretical basis for syringin to treat AD.

The BH3 interacting-domain death agonist (BID) is a pro-apoptotic member of the Bcl-2 protein family. While proteolytic processing of BID links death receptor-induced apoptosis to the mitochondrial apoptosis pathway, we previously showed that full length BID also translocates to mitochondria during Ca2+-induced neuronal cell death. Moreover, mitochondrial carrier homolog 2 (MTCH2) was identified as a mitochondrial protein that interacts with BID during cell death. We started our studies by investigating the effect of Mtch2 silencing in a well-established model of Ca2+-induced mitochondrial permeability transition pore opening in non-neuronal HCT116 cells. We found that silencing of Mtch2 inhibited mitochondrial swelling and the associated decrease in mitochondrial energetics, suggesting a pro-death function for MTCH2 during Ca2+-induced injury. Next, we explored the role of BID and MTCH2 in mediating Ca2+-induced injury in primary cortical neurons triggered by prolonged activation of NMDA glutamate receptors. Analysis of intracellular Ca2+ transients, using time-lapse confocal microscopy, revealed that neurons lacking Bid showed markedly reduced Ca2+ levels during the NMDA excitation period. These Ca2+ transients were further decreased when Mtch2 was also silenced. Collectively, our data suggest that BID and MTCH2 functionally interact to promote Ca2+-induced neuronal injury.

Acetaminophen (APAP)-induced acute liver failure (ALF) is a life-threatening disease with only a few treatment options available. Though extensive research has been conducted for more than 40 years, the underlying pathomechanisms are not completely understood. Here, we studied as to whether APAP-induced ALF can be prevented in mice by silencing the BH3-interacting domain death agonist (Bid) as a potential key player in APAP pathology. For silencing Bid expression in mice, siRNABid was formulated with the liver-specific siRNA delivery system DBTC and administered 48 h prior to APAP exposure. Mice which were pre-treated with HEPES (vehicleHEPES) and siRNALuci served as siRNA controls. Hepatic pathology was assessed by in vivo fluorescence microscopy, molecular biology, histology and laboratory analysis 6 h after APAP or PBS exposure. Application of siRNABid caused a significant decrease of mRNA and protein expression of Bid in APAP-exposed mice. Off-targets, such as cytochrome P450 2E1 and glutathione, which are known to be consumed under APAP intoxication, were comparably reduced in all APAP-exposed mice, underlining the specificity of Bid silencing. In APAP-exposed mice non-sterile inflammation with leukocyte infiltration and perfusion failure remained almost unaffected by Bid silencing. However, the Bid silencing reduced hepatocellular damage, evident by a remarkable decrease of DNA fragmented cells in APAP-exposed mice. In these mice, the expression of the pro-apoptotic protein Bax, which recently gained importance in the cell death pathway of regulated necrosis, was also significantly reduced, in line with a decrease in both, necrotic liver tissue and plasma transaminase activities. In addition, plasma levels of HMGB1, a marker of sterile inflammation, were significantly diminished. In conclusion, the liver-specific silencing of Bid expression did not protect APAP-exposed mice from microcirculatory dysfunction, but markedly protected the liver from necrotic cell death and in consequence from sterile inflammation. The study contributes to the understanding of the molecular mechanism of the APAP-induced pathogenic pathway by strengthening the importance of Bid and Bid silencing associated effects.

2-Hydroxy-3′,5,5′-trimethoxychalcone (named DK-139) is a synthetic chalcone derivative that has anti-inflammatory, anti-tumor, and endoplasmic reticulum-mediated apoptosis activities. However, the mode of action of DK-139 on reactive oxygen species (ROS)-induced apoptosis remains unknown. In this study, we found that DK-139 activated DNA damage responses, as was revealed by the accumulation of the tumor suppressor p53 and the phosphorylation of histone H2AX at Ser139 (called γ-H2AX), which are hallmarks of DNA damage responses. The occurrence of DK-139-induced DNA damage was confirmed through single-cell gel electrophoresis (comet tail assay). Interestingly, using p53-null HCT116 cells revealed that p53 was not involved in DK-139-induced apoptosis. Instead, we found that DK-139 increased the production of ROS, which led to the processing of caspase-2, BH3 interacting-domain death agonist (BID), caspase-9, and caspase-7. Pretreatment with the ROS scavenger N-acetyl cysteine reduced the frequency of DK-139-induced γ-H2AX formation, demonstrating that DK-139 triggered DNA damage through ROS production. In addition, NAC pretreatment prevented DK-139-induced processing of caspase-2, BID, caspase-9, caspase-7, and poly(ADP-ribose) polymerase. These results suggest that DK-139 triggers apoptosis through ROS-mediated DNA damage and activation of the caspase-2 cascade in A549 human lung cancer cells.

Liver tumor is difficult to cure for its high degree of malignancy and rapid progression characteristics. Ferroptosis as a new model of inducing cell death is expected to break the treatment bottleneck of liver tumors. Here, a strategy to induce ferroptosis in HepG2 cells with acid-degradable tumor targeted nanosheets Cu-Hemin-PEG-Lactose acid (Cu-Hemin-PEG-LA) is proposed. After highly ingested by HepG2 cells, Cu-Hemin-PEG-LA nanosheets are degraded by weak acid and release Cu(II) and hemin, which consuming intracellular glutathione (GSH) content and increasing the expression of heme oxygenase 1 (HMOX1) protein, respectively. Furthermore, the expression of glutathione peroxidase 4 protein (GPX4) is down-regulated by consumption intracellular GSH content via converting GSH into glutathione oxidized (GSSG), which is named the classical mode. The intracellular Fe2+ content is overloaded by the significant up-regulation of HMOX1 expression, which is denoted as nonclassical mode. The synergistic effect of classical and nonclassical mode increased the intracellular lipid reactive oxide species, induced the occurrence of ferroptosis and up-regulated the expression of BH3 interacting domain death agonist (BID), apoptosis-inducing factor (AIF), and endonuclease G proteins (EndoG). The synergistic strategy demonstrate the excellent ferroptosis induction ability and antitumor efficacy in vivo, which provides great potential for the clinical transformation of ferroptosis.

Neurodegenerative disorders such as Alzheimer���s and Parkinson���s disease as well as stroke are of growing concern in our aging societies. In the last decades, biochemical hallmarks of these pathologies, containing excessive lipid peroxide formation, iron overload and mitochondrial impairments were linked to recently described forms of regulated, oxidative cell death mechanisms such as oxytosis and ferroptosis. Neurons are vulnerable to mitochondrial damage due to their high energy demand associated with the high electrical activity. Thus, protecting mitochondria is a reasonable approach for preventing neuronal dysfunction and cell death. In particular, the pro-apoptotic protein BID is of central interest in the pathology of neurodegeneration. Studies using Bid KO mice showed a significant decrease in infarct volumes upon stroke induction. Moreover, Bid knock-out trials in HT22 neurons demonstrated that BID was crucially involved in the mechanisms of oxidative cell death model of oxytosis and ferroptosis, possibly participating in the transfer of the ROS-dependent upstream events to mitochondria. Hence, BID is an attractive target molecule for ferroptosis inhibition and, therefore, for the development of novel compounds targeting neurodegenerative diseases. However, in silico compound search was always hampered by a missing high-resolution X-ray structure of the BID protein. To improve the current understanding of the protein, the previously started approaches to elucidate the 3D crystal structure of the BID protein were continued. These experiments were conducted using the established Bid3CCSS construct. Previous high-throughput screens showed that in the Morpheus A5 condition, Bid3CCSS crystals could be consistently obtained in the form of ingrown needle clusters. Due to intensive screening of the components from the Morpheus A5 condition during the presented work, crystal quality was considerably enhanced. The best crystals from this optimization process were then subjected to a streak seeding protocol and cryoprotection. X-ray measurements of the obtained non-ingrown crystals resulted in data sets for the apo Bid3CCSS crystal with a resolution of 2.0 ��� 2.3 ��. After successful phase determination using gadolinium-acetate-soaked crystals, a crystal structure of the BID3CCSS could be modeled for the first time. The Bid3CCSS protein crystallized as a trimer. Although the protein is constructed mainly of alpha helices, large parts of two monomers were disordered, which resulted in below-average refinement statistics. However, one monomer is well-resolved and therefore could serve as a basis for targeted in silico compound development against the BID protein in the future. Further crystallographic experiments should be conducted to lower the protein���s flexibility in the crystal assembly, change the crystal packing by improving the crystal contacts, and identify (novel) binding pockets by the introduction of ligands. In the second part of the thesis, novel diphenylamine (DPA) compounds were characterized in ferroptosis. These compounds were previously developed upon a medicinal chemistry approach using the first BID inhibitor BI-6c9 as a scaffold molecule to identify novel inhibitors of oxidative cell death. The selected DPA compounds 1-3 were the most potent inhibitors against glutamate induced oxytosis in preliminary experiments. Similarly, these novel DPA compounds were very potent and selective ferroptosis inhibitors. With EC50 values between 0.23-0.32 ��M, the compounds were 10-20-fold more potent in preventing oxidative cell death than the previously described BID inhibitors. The DPA compounds abrogated lipid ROS formation, as the most conclusive hallmark of ferroptosis, as well as cytosolic and mitochondrial ROS formation without affecting the upstream cascade of ferroptosis including glutathione levels and GPX4 expression. Interestingly, a pronounced ROS scavenging effect by the DPA compounds could be excluded indicating that a direct antioxidant effect, possibly provided by the diphenylamine structure, was of minor relevance for the observed protective effects. Moreover, mitochondrial respiration and constitution were maintained in a concentration-dependent manner. Post-treatment experiments substantiated the suggestion that mitochondrial protection and an interference in the ROS development processes by the compounds may contribute to the protective mechanism. Testing BID as a potential target structure in t-BID overexpression assays, failed to demonstrate a significant effect of the DPA compounds on t-BID. Moreover, preliminary binding assays of the compounds to the Bid3CCSS protein construct using MST and NMR techniques as well as co-crystallization and soaking experiments showed no clear binding activities so far, although these assays need further optimization. In conclusion, these results highlight the role for mitochondrial protection as a strategy for the development of anti-ferroptotic compounds as possible new treatment options of neurodegenerative disorders in the future. The in vitro results encourage for further in vivo testing of the DPA compounds to evaluate the toxicity and effectiveness in models where ferroptosis might play a significant role, as for example in cerebral hemorrhage., Neurodegenerative Erkrankungen wie die Alzheimer- und Parkinson-Krankheit als auch der Schlaganfall sind von zunehmender Bedeutung in unserer alternden Gesellschaft. In den vergangenen Jahren wurden bei diesen Erkrankungsbildern biochemische Merkmale, wie eine ��berm����ige Lipidperoxidbildung, Eisen��berladung und eine Sch��digung der Mitochondrien festgestellt. Diese Merkmale sind eng mit den k��rzlich beschriebenen Formen des regulierten, oxidativen Zelltods verkn��pft, die als Oxytose und Ferroptose bezeichnet werden. F��r Neurone ist eine Sch��digung der Mitochondrien besonders problematisch, da diese infolge ihrer elektrischen Aktivit��t einen sehr hohen Energiebedarf aufweisen. Daher stellen Ans��tze, die auf den Schutz der Mitochondrien abzielen, eine sinnvolle M��glichkeit dar, neuronale Zellen vor dem Untergang zu sch��tzen. Insbesondere das pro-apoptotische Protein BID ist dabei von zentraler Bedeutung. In vitro und in vivo Studien belegen eine enge Verbindung zwischen dem BID Protein und neuronalem Zelltod, der unter anderem durch Oxytose und Ferroptose hervorgerufen sein kann. Daher ist das BID Protein ein interessantes Zielmolek��l, um z.B. Ferroptose-Inhibitoren zu entwickeln, die anschlie��end gegebenenfalls als Wirkstoffe zur Behandlung von neurodegenerativen Erkrankungen eingesetzt werden k��nnten. Die computergest��tzte in silico Suche nach neuen Wirkstoffkandidaten war jedoch bislang nur eingeschr��nkt m��glich, da hochaufgel��ste 3D-R��ntgenstrukturdaten des BID-Proteins fehlten. Daher sollte in der vorliegenden Arbeit die Kristallstruktur des BID-Proteins aufgekl��rt werden. F��r diese Experimente wurde das bereits etablierte Bid3CCSS-Konstrukt genutzt. Im Rahmen fr��herer Arbeiten durchgef��hrte Hochdurchsatz-Screens zeigten, dass in der Morpheus A5-Bedingungen zuverl��ssig Bid3CCSS Kristalle erhalten wurden, die allerdings starke Verwachsungen aufwiesen. Durch eine akribische, strukturierte Testung aller Bestandteile der Ausgangsbedingung, mit anschlie��endem Seeding und Kryo-Schutz konnte die Kristallqualit��t erheblich verbessert werden. Die durch dieses Vorgehen erhaltenen st��bchenf��rmigen Kristalle wurden anschlie��end r��ntgenkristallographisch untersucht. Dabei wurden f��r die apo Bid3CCSS-Kristalle Datens��tze mit einer Aufl��sung von 2,0-2,3 �� erhalten. Nach erfolgreicher Phasenbestimmung, mit Gadolinium-Acetat-getr��nkten Bid3CCSS Kristallen, konnte nun erstmalig eine Kristallstruktur des Bid3CCSS Proteins erstellt werden. Bid3CCSS kristallisierte dabei als Trimer. Obwohl das Protein haupts��chlich aus Alpha-Helices aufgebaut ist, lagen gr����ere Bereiche von zwei der drei Monomere weitgehend ungeordnet vor, was zu einer unterdurchschnittlichen Verfeinerungsstatistik f��hrte. Allerdings wies die Kristallstruktur eines Monomers eine definierte Elektronendichte f��r die gesamte Aminos��urenkette auf, sodass dieses f��r zuk��nftige in silico-Versuche zur Ligandenfindung herangezogen werden kann. Es wird dennoch empfohlen weiterf��hrende kristallographische Experimente durchzuf��hren, um die Flexibilit��t des Proteins im Kristallverband zu verringern, die Kristallpackung durch eine Optimierung der Kristallkontakte zu ��ndern und mit Hilfe verschiedener Ligandenfragmente neue Bindungstaschen zu identifizieren. Im zweiten Teil der Arbeit wurden neuartige Diphenylaminverbindungen (DPA) in Modellsystemen der Ferroptose charakterisiert. Diese Verbindungen wurden zuvor mittels medizinisch-chemischer Methoden entwickelt, um neuartige Inhibitoren gegen den oxidativen Zelltod zu identifizieren. Der bekannte BID-Inhibitor BI-6c9 diente dabei als Matrize. In der vorliegenden Studie zeigte sich, dass diese DPA-Verbindungen sehr potente und selektive Ferroptose-Inhibitoren darstellten. Mit EC50-Werten zwischen 0,23-0,32 ��M waren die neuen Verbindungen um das 10-20-fache protektiver gegen den oxidativen Zelltod als alle bislang beschriebenen BID-Inhibitoren. Die DPA-Verbindungen waren dabei in der Lage die Bildung von Lipid-ROS, sowie zytosolische und mitochondriale ROS Entstehung zu unterdr��cken. Diese Hemmung erfolgte ohne eine Beeinflussung der Glutathionspiegel und der GPX4-Expression, welche wesentliche Bestandteile des antioxidativen Systems der Zelle darstellen. Ausgepr��gte Radikalf��nger-Eigenschaften der DPA-Verbindungen konnten ebenfalls nicht festgestellt werden. Dahingegen wurde die mitochondriale Integrit��t durch die Verbindungen konzentrationsabh��ngig bewahrt. Nachbehandlungsexperimente untermauerten schlie��lich die Annahme, dass eine durch die DPA-Verbindungen vermittelte Mitochondrienprotektion sowie eine St��rung der ROS-Bildungsprozesse zum Schutzmechanismus beitragen. Untersuchungen der Verbindungen mittels t-BID-Toxizit��tsexperimenten, NMR- und MST-Methoden sowie Co-Kristallisations- und Soaking-Experimenten, konnten bis dato keine eindeutige Interaktion der neuen Verbindungen mit dem BID/ t-BID Protein best��tigen oder entsprechende Bindungsstellen definieren. Um diese Fragestellung allerdings abschlie��end kl��ren zu k��nnen, bed��rfen die genannten Testverfahren weiterer Optimierung. Zusammenfassend unterstreicht diese Arbeit den mitochondrialen Schutz als m��gliche Strategie f��r die Entwicklung weiterer Ferroptose-Inhibitoren, welche als zuk��nftige Behandlungsoptionen von neurodegenerativen Erkrankungen eingesetzt werden k��nnten. Die in vitro-Ergebnisse sprechen daf��r, die DPA-Verbindungen weiter in vivo zu untersuchen, um weitere Erkenntnisse zur Toxizit��t und Wirksamkeit in Modellen zu gewinnen, in denen Ferroptose eine wichtige Rolle spielen k��nnte. Daf��r w��rden sich z.B. Modelle des h��morrhagischen Schlaganfalls eignen.

In response to genotoxic stress, the tumor suppressor protein p73 induces apoptosis and cell cycle arrest. Despite extensive studies on p73-mediated apoptosis, little is known about the cytoplasmic apoptotic function of p73. Here, using H1299 lung cancer cells and diverse biochemical approaches, including colony formation, DNA fragmentation, GST pulldown, and apoptosis assays along with NMR spectroscopy, we show that p73 induces transcription-independent apoptosis via its transactivation domain (TAD) through a mitochondrial pathway and that this apoptosis is mediated by the interaction between p73-TAD and the anti-apoptotic protein B-cell lymphoma-extra large (Bcl-X(L) or BCL2L1). This binding disrupted an interaction between Bcl-X(L) and the pro-apoptotic protein BH3-interacting domain death agonist (Bid). In particular, we found that a 16-mer p73-TAD peptide motif (p73-TAD16) mediates transcription-independent apoptosis, accompanied by cytochrome c release from the mitochondria, by interacting with Bcl-X(L). Interestingly, the structure of the Bcl-X(L)–p73-TAD16 peptide complex revealed a novel mechanism of Bcl-X(L) recognition by p73-TAD. We observed that the α-helical p73-TAD16 peptide binds to a noncanonical site in Bcl-X(L), comprising the BH1, BH2, and BH3 domains in an orientation opposite to those of pro-apoptotic BH3 peptides. Taken together, our results indicate that the cytoplasmic apoptotic function of p73 is mediated through a noncanonical mode of Bcl-X(L) recognition. This finding sheds light on a critical transcription-independent, p73-mediated mechanism for apoptosis induction, which has potential implications for anticancer therapy.

Spinocerebellar ataxia type 17 (SCA17) is caused by the expansion of translated CAG repeat in the TATA box binding protein (TBP) gene encoding a long polyglutamine (polyQ) tract in the TBP protein, which leads to intracellular accumulation of aggregated TBP and cell death. The molecular chaperones act in preventing protein aggregation to ameliorate downstream harmful events. In this study, we used Tet-On cells with inducible SCA17 TBP/Q79-GFP expression to test five in-house NC009 indole compounds for neuroprotection. We found that both aggregation and polyQ-induced reactive oxygen species can be significantly prohibited by the tested NC009 compounds in Tet-On TBP/Q79 293 cells. Among the five indole compounds, NC009-1 up-regulated expression of heat shock protein family B (small) member 1 (HSPB1) chaperone to reduce polyQ aggregation and promote neurite outgrowth in neuronal differentiated TBP/Q79 SH-SY5Y cells. The increased HSPB1 thus ameliorated the increased BH3 interacting domain death agonist (BID), cytochrome c (CYCS) release, and caspase 3 (CASP3) activation which result in apoptosis. Knock down of HSPB1 attenuated the effects of NC009-1 on TBP/Q79 SH-SY5Y cells, suggesting that HSPB1 might be one of the major pathways involved for NC009-1 effects. NC009-1 further reduced polyQ aggregation in Purkinje cells and ameliorated behavioral deficits in SCA17 TBP/Q109 transgenic mice. Our results suggest that NC009-1 has a neuroprotective effect on SCA17 cell and mouse models to support its therapeutic potential in SCA17 treatment.

Eudesmin has been proven to possess anti-inflammatory effects. In the present study, the effects of eudesmin on Helicobacter pylori (H. pylori)-mediated autophagy, apoptosis, immune response and inflammation were determined in human gastric adenocarcinoma (AGS) cells in vitro and in C57BL/6 mice in vivo. Detection of the production of interleukin (IL)-8, IL-1β and immunoglobulin M (IgM) was performed using ELISA. Identification of the activation of apoptosis-associated caspase-3, -8 and -9 proteins, Bcl-2-associated X protein (Bax) and BH3 interacting domain death agonist (Bid) protein, was determined through western blot analysis. Autophagy microtubule-associated protein 1A/1B-light chain 3, isoform B (LC-3B) expression was measured using immunostaining. The results of the present study demonstrated that eudesmin inhibited the growth of H. pylori, with increased inhibition activity against antibiotic resistant strains compared with the reference strain. In addition, H. pylori-induced IL-8 secretion, LC-3B expression and apoptosis-associated protein (caspase-3, -8 and -9, Bax and Bid) activation in AGS cells was suppressed by eudesmin. Furthermore, eudesmin suppressed IL-1β and IgM production in H. pylori-infected C57BL/6 mice in vivo. In conclusion, eudesmin may be developed as a promising therapeutic agent to prevent and/or treat H. pylori-associated gastric inflammation.

Gingipains are cysteine proteases produced by Porphyromonas gingivalis, the predominant pathogen in chronic periodontitis. The present study aims to examine the role of gingipains in promoting apoptosis in osteoblasts.Human calvarial osteoblasts and osteoblast MC3T3-E1 cells were treated with 8.348 U/L gingipains. Flow cytometry analysis and terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling staining were used to detect cell apoptosis. Protein expression was examined by Western blotting, and gene expression was detected by real-time polymerase chain reaction. Small interfering (si)RNA transfection was used to knock down BH3-interacting domain death agonist (Bid) expression.Treatment with 8.348 U/L gingipains from 4 to 72 hours increased apoptosis, accompanied by elevated cleaved caspase-3 levels. Notably, gingipain-induced apoptosis was associated with increase of Bid and its truncated form, tBid, as well as p53. Transfection with Bid siRNA resulted in suppression of gingipain-induced apoptosis. The p53 inhibitor, Pifithrin-α, blocked the gingipain-induced Bid. The ability of gingipains to stimulate p53 and Bid expression was mimicked by PD-0325901 and MK-2206, the specific extracellular signal-regulated protein kinases (ERK) and protein kinase B (PKB) inhibitors, respectively. Furthermore, treatment with gingipains reduced phospho-ERK and phospho-PKB levels, an effect correlated to gingipain-induced increase in p53 and tBid expression.The present findings suggest that Bid plays an essential role in gingipain-induced osteoblast apoptosis, which is dependent on inhibition of ERK and PKB phosphorylation, followed by the activation of p53.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in the majority of tumor cells, whilst sparing normal cells. However, the potential use of TRAIL in the treatment of cancer is limited by the inevitable emergence of drug resistance. The present study reports the upregulation of latent membrane protein 1 (LMP1)-induced TRAIL resistance via the enhanced expression of X-linked inhibitor of apoptosis protein (XIAP) in nasopharyngeal carcinoma (NPC) cells. LMP1-positive NPC cells were indicated to be more sensitive to TRAIL compared with LMP1-negative NPC cells in three NPC cell lines. CNE-1 is a LMP1-negative NPC cell line that was transfected with pGL6-LMP1; following which, sensitivity to TRAIL decreased. LMP1-induced TRAIL resistance was associated with the decreased cleavage of caspase-8,-3 and -9, BH3 interacting domain death agonist (Bid) and mitochondrial depolarization, without any effects on the expression of the death receptors, B-cell lymphoma (Bcl)-2 and Bcl-extra long. Knockdown of XIAP with small interfering RNA increased caspase-3 and -9 and Bid cleavage, and prevented LMP1-induced TRAIL resistance. Furthermore, embelin, the inhibitor of XIAP, prevented LMP1-induced TRAIL resistance in the Epstein-Barr virus (EBV)-positive CNE-1-LMP1 and C666-1 NPC cell lines. However, embelin did not enhance TRAIL-induced apoptosis in NP-69, which was used as a benign nasopharyngeal epithelial cell line. These data show that LMP1 inhibits TRAIL-mediated apoptosis by upregulation of XIAP. Embelin may be used in an efficacious and safe manner to prevent LMP1-induced TRAIL resistance. The present study may have implications for the development and validation of novel strategies to prevent TRAIL resistance in EBV-positive NPC.

In all organisms, heat-shock proteins (HSPs) provide an ancient defense system. These proteins act as molecular chaperones by assisting proper folding and refolding of misfolded proteins and aid in the elimination of old and damaged cells. HSPs include Hsp100, Hsp90, Hsp70, Hsp40, and small HSPs. Through its substrate-binding domains, Hsp70 interacts with wide spectrum of molecules, ranging from unfolded to natively folded and aggregated proteins, and provides cytoprotective role against various cellular stresses. Under pathophysiological conditions, the high expression of Hsp70 allows cells to survive with lethal injuries. Increased Hsp70, by interacting at several points on apoptotic signaling pathways, leads to inhibition of apoptosis. Elevated expression of Hsp70 in cancer cells may be responsible for tumorigenesis and for tumor progression by providing resistance to chemotherapy. In contrast, inhibition or knockdown of Hsp70 reduces the size of tumors and can cause their complete regression. Moreover, extracellular Hsp70 acts as an immunogen that participates in cross presentation of MHC-I molecules. The goals of this review are to examine the roles of Hsp70 in cancer and to present strategies targeting Hsp70 in the development of cancer therapeutics.

Copper nanoparticles (Cu-NPs) have been used increasingly in various products and applications. Although recent studies have reported that exposure to Cu-NPs leads to organ accumulation and obvious toxicity, it remains unclear whether Cu-NPs can be translocated to and cause damage in the uterus. In this study, we investigated the potential for uterine injury and gene expression patterns in female rats exposed to 3.12, 6.25, or 12.5 mg/kg/d Cu-NPs via intraperitoneal injection for 14 consecutive days. The results indicated that exposure to Cu-NPs led to significant decreases in the relative uterine weight coefficients and increases in inflammatory cell infiltration, mitochondrial swelling and vacuolization, shortened and reduced endometrial epithelial cell microvilli, and apoptosis. Furthermore, exposure to Cu-NPs increased malondialdehyde (MDA) accumulation and decreased superoxide dismutase (SOD) levels. Signal transduction mechanism studies indicated that exposure to Cu-NPs activated caspases 3, 8, and 9 and BH3 interacting domain death agonist (tBid), reduced B cell leukemia/lymphoma 2 (Bcl-2) expression, and increased the expression of apoptotic peptidase activating factor 1 (Apaf-1), BCL2-associated X, apoptosis regulator (Bax), and cytochrome c. A microarray analysis revealed significant alterations in the expression of 963 genes; of these, 622 were upregulated and 341 were downregulated. The results of further evaluations of some altered genes, including matrix metallopeptidase 12 (Mmp12), using quantitative RT-PCR agreed with the microarray findings. These results provide strong evidence that Cu-NPs can trigger both intrinsic and extrinsic apoptotic pathways to mediate uterine injury, resulting in oxidative stress-related changes in gene expression.

Aim Arsenic trioxide (As2O3), known as pi-shuang and the most toxic compound in traditional Chinese medicine, has been used as an antitumor agent for thousands of years. Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a natural phenol that has significant anti-bacterial, anti-fungaI and antiaging activities. Our study aimed to examine the combined anticancer effects of As2O3 and resveratrol against human neuroblastoma SK-N-SH cells, and elucidate the underlying intracellular signaling. Materials and methods SK-N-SH cells were treated with an extremely low-dose (2-4 μM) of As2O3 alone or combined with 75 μg/ml resveratrol for further comparisons. Cell viability, apoptotic signaling as well as synergistic cytotoxic effects were estimated using the MTT assay, microscopy observation, flow cytometric analysis for loss of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS), and typical quantitative western blotting analysis. Student's t-test, and one- and two-way analysis of variance (ANOVA) were used for examination of significant differences. Results The combined treatment was more effective than single treatment of As2O3 or resveratrol alone in suppressing cell viability, which correlated with the elevation of ROS levels. The intracellular mechanisms of cytotoxicity of As2O3 plus resveratrol were revealed as ROS accumulation and relative decrease of MMP, leading to activation of caspase-3 and -9, but not of caspase-1, -7 and-8. Combination treatment reduced the expression of B-cell lymphoma 2 (BCL2), BH3 interacting domain death agonist (BID), and BCL-x/L. Conclusion Combined treatment at extremely low concentration of two agents from natural products, As2O3 and resveratrol, has high potential as a cocktail of anticancer drugs for neuroblastoma.

Su Yang Decoction (SYD) is a popular healthcare product comprised of various brassicaceous vegetables known to exhibit anticancer activity. However, the effects of SYD on tumor growth, following its combination with brassicaceous vegetables into a compound formula, require further investigation. In the present study, a quality control of SYD was subjected to high-performance liquid chromatography for the quantitative and qualitative determination of sulforaphane, the main anticancer component of SYD. SYD inhibited colon cancer cell proliferation in a dose- and time-dependent manner and induced G1 phase arrest in colon cancer HT-29 cell lines. In addition, SYD triggered caspase-mediated apoptosis in a dose-dependent manner and induced the cleavage of poly (ADP-ribose) polymerase, tumor necrosis factor superfamily member 10, X-linked inhibitor of apoptosis, and truncated BH3 interacting domain death agonist. Furthermore, the expression of FADD-like interleukin-1β-converting enzyme (FLICE)-like inhibitory proteins (FLIPs) and long isoform of FLICE-inhibitory protein was reduced by SYD and the direct targeting of cellular-FLIP with small interfering RNA inhibited colon cancer cell proliferation and decreased the SYD concentration required for proliferation inhibition. SYD treatment was also associated with the translocation of proapoptotic BCL2 associated X, apoptosis regulator to the mitochondria and the release of cytochrome c from the mitochondria to the cytosol. The aforementioned results indicate that SYD exerts anti-colorectal cancer effects through an underlying mechanism that may involve caspase activation.

Avian ciliary ganglion (CG) development involves a transient execution phase of apoptosis controlling the final number of neurons, but the time-dependent molecular mechanisms for neuronal cell fate are largely unknown. To elucidate the molecular networks regulating important aspects of parasympathetic neuronal development, a genome-wide expression analysis was performed during multiple stages of avian CG development between embryonic days E6 and E14. The transcriptome data showed a well-defined sequence of events, starting from neuronal migration via neuronal fate cell determination, synaptic transmission, and regulation of synaptic plasticity to growth factor associated signaling. In particular, we extracted a neuronal apoptosis network that characterized the cell death execution phase at E8/E9 and apoptotic cell clearance at E14 by combining the gene time series analysis with network synthesis from the chicken interactome. Network analysis identified TP53 as key regulator and predicted involvement of the BH3 interacting domain death agonist (BID). A virus-based RNAi knockdown approach in vivo showed a crucial impact of BID expression on the execution of ontogenetic programmed cell death (PCD). In contrast, Bcl-XL expression did not impact PCD. Therefore, BID-mediated apoptosis represents a novel cue essential for timing within CG maturation.

Neutrophils are key players in the early defense against invading pathogens. Due to their potent effector functions, programmed cell death of activated neutrophils has to be tightly controlled; however, its underlying mechanisms remain unclear. Fas ligand (FASL/CD95L) has been shown to induce neutrophil apoptosis, which is accelerated by the processing of the BH3-only protein BH3 interacting domain death agonist (BID) to trigger mitochondrial apoptotic events, and been attributed a regulatory role during viral and bacterial infections. Here, we show that, in accordance with previous works, mouse neutrophils underwent caspase-dependent apoptosis in response to FASL, and that this cell death was significantly delayed upon loss of BID. However, pan-caspase inhibition failed to protect mouse neutrophils from FASL-induced apoptosis and caused a switch to RIPK3-dependent necroptotic cell death. Intriguingly, such a switch was less evident in the absence of BID, particularly under inflammatory conditions. Delayed neutrophil apoptosis has been implicated in several auto-inflammatory diseases, including inflammatory bowel disease. We show that neutrophil and macrophage driven acute dextran sulfate sodium (DSS) induced colitis was slightly more aggravated in BID-deficient mice, based on significantly increased weight loss compared to wild-type controls. Taken together, our data support a central role for FASL > FAS and BID in mouse neutrophil cell death and further underline the anti-inflammatory role of BID.

Background: Diabetic nephropathy is the most common cause of end-stage renal disease. Emerging evidences indicate that many mechanistic pathways including apoptosis play an important role in the pathogenesis and progression of macrovascular and microvascular complications of diabetes mellitus. The aim of the present study is to show the effects of grape seed extract (GSE) on oxidative stress and apoptosis in the kidney of streptozotocin-induced diabetic rats. Materials and methods: The study included control group, diabetic group without treatment and diabetic group treated with GSE (n=7) group. GSE was given orally (100 mg/kg/day) for six weeks. Following parameters were evaluated; oxidative stress index, caspase 1, IL1-alpha, caspase 2, IL1-beta, BCL2-associated agonist of cell death (BAD), X-linked inhibitor of apoptosis (XIAP), DNA fragmentation factor, alpha subunit and beta bubunit (DFFA, DFFB), BH3 interacting domain death agonist (BID), caspase 6, Bcl2-like 1 (BCL-XL), caspase 8, tumor necrosis factor receptor superfamily, member 1 b (TNFRSF1B) and IAP-binding mitochondrial protein (DIABLO). Results: Oxidative stress index levels were significantly increased in the kidney of diabetic group without treatment compared to control group, and decreased in diabetic + GSE group compared to diabetic group without treatment. In the kidney of diabetic group without treatment, caspase 1, IL-1 alpha, BAD, DFFA, DFFB and caspase-6 gene expressions were significantly higher compared to control group. In diabetic + GSE group caspase 1, caspase 2, XIAP, DFFA, BID, BCL-XL and TNFRSF1B genes were significantly decreased compared to control group. Conclusions: Grape seed reduces oxidative stress and apoptosis gene expression suggesting the protective effect on diabetic nephropathy. © 2015 Informa Healthcare USA, Inc. All rights reserved: reproduction in whole or part not permitted

Cadmium (Cd) is a toxic metal capable of damaging brain. Studies have demonstrated that Cd can induce apoptosis in neuronal cells. The CD95/APO-1 (Fas)/Fas Ligand (FasL) signaling pathway is one of the primary apoptosis pathways, but the role and regulatory mechanism of this pathway in neuronal cells remain unclear. Here, we demonstrated the underlying mechanism of the Fas/FasL system involving the mitochondrial apoptotic pathway in neuronal cells. Primary rat cerebral cortical neurons and PC12 cells were exposed to Cd, which significantly activated expression of Fas, FasL, Fas-associated death domain (FADD) and cleaved caspase-8. However, expression of cleaved caspase-8 decreased at 20 µM Cd in primary cerebral cortical neurons. Importantly, Cd-induced apoptotic morphological changes and increase in the apoptosis rate were partially blocked by Z-IETD-FMK, which is a specific inhibitor of caspase-8. Cd-mediated increase of apoptosis rate was inhibited by anti-FasL antibody. Furthermore, our data revealed that Z-IETD-FMK also blocked increase of truncated BH3 interacting domain death agonist (tBID)/BID, decrease of the B-cell lymphoma 2 (Bcl-2)/Bcl-2 associate X protein (Bax) ratio and mitochondrial membrane potential (MMP), release of cytochrome c, as well as cleavage of caspase-9/3 and poly (ADP-ribose) polymerase (PARP) induced by Cd. Taken together, our results demonstrate that the Fas/FasL-mediated mitochondrial apoptotic pathway plays an important role in Cd-induced neuronal apoptosis.

Anti-GPIIb/IIIa-mediated complement activation has been reported to be important in the pathogenesis of immune thrombocytopenia (ITP). However, the role of the complement system and the involved regulatory mechanism remain equivocal. Beta2-glycoprotein I (β2-GPI), known as the main target for antiphospholipid autoantibodies, has been demonstrated as a complement regulator. Here, we investigated the complement-regulatory role of β2-GPI in anti-GPIIb/IIIa-mediated ITP. Plasma complement activation and enhanced complement activation capacity (CAC) were found in ITP patients with anti-GPIIb/IIIa antibodies in vivo and in vitro. Diminished plasma levels of β2-GPI were shown in patients of this group, which was inversely correlated with C5b-9 deposition. C5b-9 generation was inhibited by approximate physiological concentrations of β2-GPI, in a dose-dependent manner. Inhibition of C3a generation by β2-GPI and the existence of β2-GPI/C3 complexes in plasma indicated a regulation on the level of the C3 convertase. Furthermore, β2-GPI down-regulated the phosphorylation levels of c-Jun N-terminal kinase (JNK) and cleavage of BH3 interacting domain death agonist (Bid) and ultimately harbored platelet lysis. Our findings may provide a novel link between diminished plasma levels of β2-GPI and enhanced complement activation, indicating β2-GPI as a potential diagnostic biomarker and therapeutic target in the treatment of anti-GPIIb/IIIa-mediated ITP.

Ischemic postconditioning has been demonstrated to alleviate brain ischemia/reperfusion-induced neuronal apoptosis; however, the protective mechanisms underlying the improved and more convenient method of remote ischemic postconditioning (RIPostC) are only recently beginning to be elucidated. Mitochondria are important in the regulation of cell apoptosis, and the B‑cell lymphoma 2 (Bcl‑2) homology 3 interacting‑domain death agonist (BID) promotes the insertion/oligomerization of Bcl‑2‑associated X protein into the mitochondrial outer membrane, leading to the release of proapoptotic proteins from the mitochondria. The present study hypothesized that RIPostC targets the BID‑mediated mitochondrial apoptotic pathway to exert neuroprotective effects, and the optimal time window for RIPostC application was investigated. RIPostC was conducted as follows: Three 10‑min cycles of bilateral femoral artery occlusion with intervals of 10 min reperfusion after 0, 10 or 30 min of brain reperfusion. The results revealed that reperfusion induced significant activation of BID, via proteolytic cleavage and translocation to the mitochondria, as determined using western blot analysis and immunofluorescence staining. Mitochondrial release of cytochrome c was additionally detected during BID activation, all of which were inhibited by the application of RIPostC. When RIPostC was applied during reperfusion, it demonstrated a significant protective effect. Furthermore, the infarct volume, neurological function and the degree of neuronal apoptosis were improved with application of RIPostC. These results suggested that the protective mechanisms of RIPostC may be associated with inhibition of the BID‑mediated mitochondrial apoptotic pathway, which may act as a potential molecular target for therapeutic intervention in the future.

Previously we have shown that interferon (IFN)-α induced apoptosis is predominantly mediated by the upregulation of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) via the caspase-8 pathway. It was also shown that recruitment of mitochondria in IFN-α induced apoptosis involves the cleavage of BH3 interacting domain death agonist (Bid) to truncated Bid (tBid). In the present study, we demonstrate that tBid induced by IFN-α2a activates mitochondrial Bak to trigger the loss of mitochondrial membrane integrity, consequently causing release of apoptosis-inducing factor (AIF) in ovarian cancer cells, OVCAR3. AIF translocates from the mitochondria to the nucleus and induces nuclear fragmentation and cell death. Both a small molecule Bid inhibitor (BI-6C9) or Bid-RNA interference (RNAi) preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated IFN-α2a-induced cell death. Cell death induced by tBid was inhibited by AIF-RNAi, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that BI-6C9 did not prevent the release of cytochrome c from mitochondria to cytosol, while the release of AIF was prevented. In conclusion, IFN-α2a-induced apoptosis is mediated via the mitochondria-associated pathway involving the cleavage of Bid followed by AIF release that involves Bak activation and translocation of AIF from the mitochondria to the nucleus in OVCAR3 cells.

The PIDDosome, a multiprotein complex constituted of the 'p53-induced protein with a death domain (PIDD), 'receptor-interacting protein (RIP)-associated ICH-1/CED-3 homologous protein with a death domain' (RAIDD) and pro-Caspase-2 has been defined as an activating platform for this apoptosis-related protease. PIDD has been implicated in p53-mediated cell death in response to DNA damage but also in DNA repair and nuclear factor kappa-light-chain enhancer (NF-κB) activation upon genotoxic stress, together with RIP-1 kinase and Nemo/IKKγ. As all these cellular responses are critical for tumor suppression and deregulated expression of individual PIDDosome components has been noted in human cancer, we investigated their role in oncogenesis induced by DNA damage or oncogenic stress in gene-ablated mice. We observed that Pidd or Caspase-2 failed to suppress lymphoma formation triggered by γ-irradiation or 3-methylcholanthrene-driven fibrosarcoma development. In contrast, Caspase-2 showed tumor suppressive capacity in response to aberrant c-Myc expression, which did not rely on PIDD, the BH3-only protein Bid (BH3 interacting domain death agonist) or the death receptor ligand Trail (TNF-related apoptosis-inducing ligand), but associated with reduced rates of p53 loss and increased extranodal dissemination of tumor cells. In contrast, Pidd deficiency associated with abnormal M-phase progression and delayed disease onset, indicating that both proteins are differentially engaged upon oncogenic stress triggered by c-Myc, leading to opposing effects on tumor-free survival.

TNF@a-mediated apoptosis is one of the complex and tightly regulated cellular processes as it involves the activation of both pro- and anti-apoptotic signaling pathways. Thus, it is important to elucidate the molecular players of this process and their dynamics in order to gain an in-depth understanding of the mechanisms underlying apoptosis. To this end, we proposed an integrated model of TNF@a-mediated apoptosis pathway in Type I cells, formulated based on the principles of mass action kinetics. The model includes major apoptotic modules-the extrinsic and intrinsic pathways, the NF@kB survival signaling and various regulatory mechanisms. We performed simulations and sensitivity analyses to study the role of NF@kB pathway in regulating apoptosis, and identified IAP as one of the more potent regulators of apoptosis.

The avian influenza virus H9N2 subtype has circulated in wild birds, is prevalent in domestic poultry, and has successfully crossed the species boundary to infect humans. Phylogenetic analyses showed that viruses of this subtype appear to have contributed to the generation of highly pathogenic H5N1 viruses. Little is known about the host responses to H9N2 viruses in human airway respiratory epithelium, the primary portal for viral infection. Using an apically differentiated primary human tracheobronchial epithelial (TBE) culture, we examined host immune responses to infection by an avian H9N2 virus, in comparison with a human H9N2 isolate. We found that IFN-β was the prominent antiviral component, whereas interferon gamma-induced protein 10 kDa (IP-10), chemokine (C-C motif) ligand (CCL)-5 and TNF-α may be critical in proinflammatory responses to H9N2 viruses. In contrast, proinflammatory IL-1β, IL-8, and even IL-6 may only play a minor role in pathogenicity. Apparently Toll-like receptor (TLR)-3, TLR-7, and melanoma differentiation- associated gene 5 (MDA-5) contributed to the innate immunity against theH9N2viruses, andMDA-5was important in the induction of IFN-β. We showed that the avian H9N2 virus induced apoptosis through the mitochondria/cytochrome c-mediated intrinsic pathway, in addition to the caspase 8-mediated extrinsic pathway, as evidenced by the cytosolic presence of active caspase 9 and cytochrome c, independent of truncated BH3 interacting domain death agonist (Bid) activation. Further, we demonstrated that FLICE-like inhibitory protein (FLIP), an apoptotic dual regulator, and the p53- dependent Bcl-2 family members, Bax and Bcl-x s, appeared to be involved in the regulation of extrinsic and intrinsic apoptotic pathways, respectively. The findings in this study will further our understanding of host defense mechanisms and the pathogenesis of H9N2 influenza viruses in human respiratory epithelium., link_to_subscribed_fulltext

Apoptosis, an essential and basic biological phenomenon, is regulated in a complex manner by a multitude of factors. Myeloid cell leukemia 1 (Mcl-1), an anti-apoptotic member of the B-cell lymphoma 2 (Bcl-2) family of apoptosis-regulating proteins, exemplifies a number of the mechanisms by which a protein’s contribution to cell fate may be modified. The N-terminus of Mcl-1 is unique amongst the Bcl-2 family, in that it is rich in experimentally confirmed and putative regulatory residues and motifs. These include sites for ubiquitination, cleavage and phosphorylation, which influence the protein’s stability, localisation, dimerization and function. Here we review what is known about the regulation of Mcl-1 expression and function, with particular focus on post-translational modifications and how phosphorylation interconnects the complex molecular control of Mcl-1 with cellular state.

Prion protein (PrPc) was originally viewed solely as being involved in prion disease, but now several intriguing lines of evidence have emerged indicating that it plays a fundamental role not only in the nervous system, but also throughout the human body. PrPc is expressed most abundantly in the brain, but has also been detected in other non-neuronal tissues as diverse as lymphoid cells, lung, heart, kidney, gastrointestinal tract, muscle, and mammary glands. Recent data indicate that PrPc may be implicated in biology of glioblastoma, breast cancer, prostate and gastric cancer. Over expression of PrPc is correlated to the acquisition by tumor cells of a phenotype for resistance to cell death induced by TNF alpha and TRAIL or antitumor drugs such as paclitaxel and anthracyclines. PrPc may promote tumorigenesis, proliferation and G1/S transition in gastric cancer cells. This review revisits the physiological functions of PrPc, and its possible implications for cancer biology.

BH3 interacting domain death agonist (Bid), a pro-apoptotic member of the Bcl-2 family of proteins, is activated through cleavage by caspase-8. The active C-terminal fragment of Bid (tBid) translocates to the mitochondria where it interacts with cardiolipins at contact sites and induces the release of cytochrome c by a mechanism that is not yet fully understood. It has been shown that the alpha-helices αH6 and αH7 (which create the hairpin-forming domain of tBid) mediate the insertion of Bid into mitochondrial membranes and are essential for the cytochrome c-releasing activity. In the present study, we focused on the interaction between the αH6 and the mitochondrial membrane. By the use of single-cell electropermeabilization associated with flow cytometric analysis of intact cells, we demonstrated that H6 is able to induce cell death when used in the micromolar range. We also studied the interactions of the αH6 with artificial monolayers. We showed that the presence of negatively charged cardiolipins greatly enhances the insertion of αH6 into the phospholipid monolayer. The modification of two charged amino acid residues in αH6 abolished its insertion and also its in vivo effects. Furthermore, the negative values of the excess areas of mixing indicate that attractive interactions between cardiolipins and αH6 occur in the mixed monolayers. Fluorescence microscopy observations revealed that αH6 significantly disrupts cardiolipin packing and stabilizes the fluid lipid phase. These results suggest that cardiolipins at the contact sites between the two mitochondrial membranes could mediate the binding of tBid via αH6.

FAS (also called APO-1 and CD95) and its physiological ligand, FASL, regulate apoptosis of unwanted or dangerous cells, functioning as a guardian against autoimmunity and cancer development. Distinct cell types differ in the mechanisms by which the 'death receptor' FAS triggers their apoptosis. In type I cells, such as lymphocytes, activation of 'effector caspases' by FAS-induced activation of caspase-8 suffices for cell killing, whereas in type II cells, including hepatocytes and pancreatic beta-cells, caspase cascade amplification through caspase-8-mediated activation of the pro-apoptotic BCL-2 family member BID (BH3 interacting domain death agonist) is essential. Here we show that loss of XIAP (X-chromosome linked inhibitor of apoptosis protein) function by gene targeting or treatment with a second mitochondria-derived activator of caspases (SMAC, also called DIABLO; direct IAP-binding protein with low pI) mimetic drug in mice rendered hepatocytes and beta-cells independent of BID for FAS-induced apoptosis. These results show that XIAP is the critical discriminator between type I and type II apoptosis signalling and suggest that IAP inhibitors should be used with caution in cancer patients with underlying liver conditions.

Sulforaphane (SFN) is a naturally occurring chemopreventive agent, which effectively inhibits proliferation of HepG2 human hepatocellular carcinoma cells via mitochondria‑mediated apoptosis. Endoplasmic reticulum stress is considered the most important cause of cell apoptosis; therefore, the present study aimed to determine whether the endoplasmic reticulum pathway was involved in SFN-induced apoptosis of HepG2 cells. An MTT assay was used to detect the inhibitory effects of SFN on HepG2 cells. Fluorescence microscopy was used to observe the morphological changes in apoptotic cells, and western blot analysis was conducted to detect the expression of binding immunoglobulin protein (Bip)/glucose-regulated protein 78 (GRP78), X‑box binding protein‑1 (XBP‑1) and BH3 interacting domain death agonist (Bid). Furthermore, flow cytometry was used to determine the apoptotic rate of HepG2 cells, and the protein expression of C/EBP homologous protein (CHOP)/growth arrest‑ and DNA damage‑inducible gene 153 (GADD153) and caspase-12 in HepG2 cells. The results indicated that SFN significantly inhibited the proliferation of HepG2 cells; the half maximal inhibitory concentration values were 32.03±0.96, 20.90±1.96 and 13.87±0.44 µmol/l, following treatment with SFN for 24, 48 and 72 h, respectively. Following 48 h of SFN treatment (10, 20 and 40 µmol/l), the apoptotic rates of HepG2 cells were 31.8, 61.3 and 77.1%, respectively. Furthermore, after 48 h of exposure to SFN, the cells presented typical morphological alterations of apoptosis, as detected under fluorescence microscopy. Treatment with SFN for 48 h also significantly upregulated the protein expression levels of Bip/GRP78, XBP‑1, caspase‑12, CHOP/GADD153 and Bid in HepG2 cells. In conclusion, endoplasmic reticulum stress may be considered the most important mechanism underlying SFN-induced apoptosis in HepG2 cells.

Acute kidney injury is a clinical syndrome characterized by a loss of renal function and acute tubular necrosis. Resveratrol exerts a wide range of pharmacological effects based on its anti‑inflammatory, antioxidant and cytoprotective properties. The present study aimed to evaluate whether resveratrol attenuates acute kidney injury in a cisplatin‑induced rat model and to investigate the potential mechanisms involved. Rats were randomly divided into four treatment groups: control, cisplatin, resveratrol, and cisplatin plus resveratrol. Rats exposed to cisplatin displayed acute kidney injury, identified by analysis of renal function and histopathological observation. Resveratrol significantly ameliorated the increased serum creatinine, blood urea nitrogen, renal index and histopathological damage induced by cisplatin. Furthermore, compared with untreated control animals, cisplatin lead to significantly increased expression of Fas ligand, tumor necrosis factor‑α (TNF‑α), caspase‑8 and Bcl‑2 associated protein X apoptosis regulator (Bax), and decreased expression of anti‑apoptosis regulators, BH3 interacting domain death agonist (BID) and B cell lymphoma 2 apoptosis regulator (Bcl‑2). Administration of resveratrol significantly reversed the cisplatin‑induced alteration in these apoptosis‑associated proteins. In conclusion, these findings suggest that resveratrol attenuates cisplatin‑induced acute kidney injury through inactivation of the death receptor‑mediated apoptotic pathway, and may provide a new therapeutic strategy to ameliorate the process of acute kidney injury.

Macroautophagy, which will hereafter be referred to as autophagy, is an evolutionarily conserved process, during which cells recycle and remove damaged organelles and proteins in response to cellular stress. However, the mechanisms underlying the regulation of autophagy remain to be fully elucidated. The present study demonstrated that knockdown of zinc finger protein like 1 (ZFPL1) induces autophagy and increases autophagic cell death in NCI‑N87 and BGC‑823 human gastric carcinoma cell lines. To examine the role of ZFPL1 in gastric carcinoma cells, ZFPL1 expression was downregulated by lentiviral infection. Zinc finger domain‑FLAG was used to compete with ZFPL1 for golgin A2/GM130 binding. Autophagy was analyzed by red fluorescent protein‑microtubule‑associated protein 1A/1B‑light chain 3 (LC3) puncta, LC3I to LC3II conversion, and p62 expression. The results demonstrated that knockdown of ZFPL1 was able to significantly increase cell death rate. However, ZFPL1 knockdown exerted almost no effect on the expression of apoptosis‑associated markers, including B cell lymphoma 2 (Bcl‑2), Bcl‑x, Bcl‑2‑associated X protein, BH3 interacting domain death agonist, p53, and the classical caspase family members, caspase‑3, caspase‑8 and caspase‑9. An endogenous ZFPL1‑GM130 association was identified in NCI‑N87 cells and BGC‑823 cells by co‑immunoprecipitation. Furthermore, cell death was restricted following treatment of ZFPL1 knockdown cells with an autophagy inhibitor. Therefore, knockdown of ZFPL1 expression may induce cell death via autophagy, rather than apoptosis. These results suggest that ZFPL1 may serve an important role in regulating autophagy in NCI‑N87 and BGC‑823 cells.

Glucocorticoid-induced leucine zipper (GILZ) is a potent anti-inflammatory protein, the expression of which is mainly induced by glucocorticoids (GCs) in haematopoietic cells. GILZ regulates signal transduction pathways of inflammation and plays a role in cell survival. The objective of this study was to evaluate the expression and mechanisms of action of GILZ in the apoptosis of human neutrophils. GILZ expression was induced by GCs in human neutrophils, enhanced upon phosphatidylinositol 3-kinase inhibition and resulted in apoptosis amplification. We then stably transfected PLB-985 cells with the human gilz gene and differentiated both control and GILZ-overexpressing clones in neutrophil-like cells. GILZ overexpression in PLB-985 cells led to an exacerbated apoptosis, associated with caspase-3, caspase-9 and caspase-8 activations, and a loss of mitochondrial potential, suggesting that GILZ-induced apoptosis used the mitochondrial pathway. The expression of BH3 interacting domain death agonist, Bcl-2 interacting mediator of cell death, annexin-A1 and Bcl-2-associated X was not affected in PLB-985-GILZ clones, but phosphorylation and subsequent proteasomal degradation of myeloid cell leukemia-1 (Mcl-1) were observed. Noteworthy, Mcl-1 phosphorylation was related to a significant and sustained activation of c-Jun N-terminal kinase (JNK) in PLB-985-GILZ clones. These results reveal GILZ to be a new actor in apoptosis regulation in neutrophil-like cells involving JNK and Mcl-1.

Bidens pilosa, a medicinal herb worldwide, is rich in bioactive polyynes. In this study, by using high resolution 2-dimensional gel electrophoresis coupled with mass spectrometry analysis, as many as 2000 protein spots could be detected and those whose expression was specifically up- or downregulated in Jurkat T cells responsive to the treatment with 2-β-D-glucopyranosyloxy-1-hydroxytrideca-5,7,9,11-tetrayne (GHTT) can be identified. GHTT treatment can upregulate thirteen proteins involved in signal transduction, detoxification, metabolism, energy pathways, and channel transport in Jurkat cells. Nine proteins, that is, thioredoxin-like proteins, BH3 interacting domain death agonist (BID protein involving apoptosis), methylcrotonoyl-CoA carboxylase beta chain, and NADH-ubiquinone oxidoreductase, were downregulated in GHTT-treated Jurkat cells. Further, bioinformatics tool, Ingenuity software, was used to predict signaling pathways based on the data obtained from the differential proteomics approach. Two matched pathways, relevant to mitochondrial dysfunction and apoptosis, in Jurkat cells were inferred from the proteomics data. Biochemical analysis further verified both pathways involving GHTT in Jurkat cells. These findings do not merely prove the feasibility of combining proteomics and bioinformatics methods to identify cellular proteins as key players in response to the phytocompound in Jurkat cells but also establish the pathways of the proteins as the potential therapeutic targets of leukemia.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease and one of the cancer entities with the lowest life expectancy. Beside surgical therapy, no effective therapeutic options are available yet. Here, we show that 4-phenylbutyrate (4-PB), a known and well-tolerable inhibitor of histone deacetylases (HDAC), induces up to 70% apoptosis in all cell lines tested (Panc 1, T4M-4, COLO 357, BxPc3). In contrast, it leads to cell cycle arrest in only half of the cell lines tested. This drug increases gap junction communication between adjacent T3M-4 cells in a concentration-dependent manner and efficiently inhibits cellular export mechanisms in Panc 1, T4M-4, COLO 357 and BxPc3 cells. Consequently, in combination with gemcitabine 4-PB shows an overadditive effect on induction of apoptosis in BxPc3 and T3M-4 cells (up to 4.5-fold compared to single drug treatment) with accompanied activation of Caspase 8, BH3 interacting domain death agonist (Bid) and poly (ADP-ribose) polymerase family, member 1 (PARP) cleavage. Although the inhibition of the mitogen-activated protein kinase-pathway has no influence on fulminant induction of apoptosis, the inhibition of the JNK-pathway by SP600125 completely abolishes the overadditive effect induced by the combined application of both drugs, firstly reported by this study.

Caspase-independent cell death may have a critical role to play in the therapeutic destruction of tumours. Recently it has been suggested that one of the mechanisms by which rituximab, a therapeutic anti-CD20 antibody, kills B cells is caspase-independent. In this study we show that rituximab can induce death in a variety of Burkitt lymphoma derived cell lines. Rituximab-treated cells show leakage of adenylate kinase, surface expression of phosphatidylserine, upregulation of the cellular stress protein HSP70, phosphorylation of the survival protein Akt, and depolarisation of the mitochondrial membrane but no loss of cytochrome c or apoptosis inducing factor. Caspase inhibitors do not block these events. In support of these data there is no cleavage of caspases 3, 8 and 9, poly(ADP-ribose) polymerase, BH3 interacting domain death agonist or genomic DNA. Morphologically, cells show nuclear enlargement and cytoplasmic vacuolisation. Triggering of receptor mediated death in CD95 responsive lines results in "classical" apoptosis indicating that the internal machinery necessary for apoptosis is intact in these lines. The results suggest that rituximab can kill human B cells via a caspase-independent form of programmed cell death that shares features of apoptosis and necrosis. This pathway may be relevant to the clinical efficacy of rituximab.

Background: Two main pathways of Fas-mediated apoptosis have been identified: the Type-I (death-inducing signaling complex) pathway and the Type-II (mitochondrial) pathway. While apoptotic cell death has been implicated in lumbar degenerative disc disease, we are not aware of any studies in which surgically removed discs from live humans have been examined to determine which of the two pathways is involved in the apoptosis of disc cells. As an initial step in the development of therapies to inhibit inappropriate or premature apoptosis of disc cells, our objective was to determine which pathway is involved. Methods: We examined thirty-two samples of herniated lumbar disc tissue with use of immunohistochemical staining and Western blot analysis to determine the presence of several proteins, including caspase-8 (associated with the Type-I pathway); BID (BH3 interacting domain death agonist), cytochrome-c, and caspase-9 (associated with the Type-II pathway); and caspase-3 (an executioner of apoptosis). The TUNEL (terminal deoxynucleotidyl transferase [TDT]-mediated dUTP nick end labeling) assay was performed to confirm the occurrence of apoptosis of the disc cells. Results: The proteins associated with the Type-II pathway (BID, cytochrome-c, and caspase-9) stained positively in all samples. Although the protein associated with the Type-I pathway (caspase-8) was not detected on immunohistochemical analysis, a small amount of caspase-8 was detected on Western blot analysis. However, the expression of Type-II proteins was still higher than the expression of caspase-8 on Western blot analysis. The expression of caspase-3 was identified in all samples with immunohistochemical and Western blot analysis. TUNEL-positive disc cells were identified in all samples. Conclusions: The results of the present study suggest that human disc cells are Type-II cells, which undergo apoptotic cell death through mitochondrial involvement. Clinical Relevance: Future therapeutic modalities to inhibit inappropriate or premature apoptotic cell death in degenerating discs should be directed to the mitochondrial pathway.

Hepatitis C virus (HCV) encodes a polyprotein consisting of core, envelope (E1, E2, p7), and nonstructural polypeptides (NS2, NS3, NS4A, NS4B, NS5A, NS5B). The serine protease (NS3/NS4A), helicase (NS3), and polymerase (NS5B) constitute valid targets for antiviral therapy. We engineered BH3 interacting domain death agonist (BID), an apoptosis-inducing molecule, to contain a specific cleavage site recognized by the NS3/NS4A protease. Cleavage of the BID precursor molecule by the viral protease activated downstream apoptotic molecules of the mitochondrial pathway and triggered cell death. We extended this concept to cells transfected with an infectious HCV genome, hepatocytes containing HCV replicons, a Sindbis virus model for HCV, and finally HCV-infected mice with chimeric human livers. Infected mice injected with an adenovirus vector expressing modified BID exhibited HCV-dependent apoptosis in the human liver xenograft and considerable declines in serum HCV titers.

Pro-apoptotic members of the Bcl-2 family can be subdivided in two classes according to their structure: a group including Bax, Bak, and Bok that display Bcl-2 homology (BH) 1, BH2 and BH3 domains and a second group including Bid (BH3 interacting domain death agonist), Bad, Bim (Bcl-2 interacting mediator of cell death) and several others that contain only a BH3 domain, the BH3-only proteins. The BH3-only proteins have been proposed to activate pro-apoptotic members of the Bax subfamily to trigger a mitochondrial pathway that leads to the release of cytochrome c and other apoptogenic factors. Here we report that the mechanism of action of Bim is different from that of Bid. Although overexpression of Bid or Bim in cells leads to cytochrome c release, only Bid is able to trigger the release of cytochrome c through Bax activation when added directly to isolated mitochondria. BimL, although unable to activate Bax, can directly inhibit Bcl-2 or Bcl-xL. Our data suggest two functional classes of BH3-only proteins: those such as Bid which directly activate Bax-like proteins leading to mitochondrial membrane permeability and apoptosis and those such as Bim which inhibit anti-apoptotic proteins and render the cells more susceptible to apoptogenic stimuli.

Background Endoplasmic reticulum (ER) stress and autophagy each play important roles in hepatocyte cell injury. We hypothesized that gene expression of C/EBP-homologous protein (CHOP) and the BH3 proteins Bcl2-interacting mediator of cell death (BIM) and BH3-interacting domain death agonist (BID) are involved in a complex interplay that regulates ER stress-induced autophagy and cell death. Materials and methods Hepatocytes were cultured from lean Zucker rats. Confluent hepatocytes were incubated with single or combined small interfering RNA for CHOP, BIM, and/or BID for 24 h providing gene inhibition. Incubation with tunicamycin (TM) for another 24 h stimulated ER stress. Quantitative real-time polymerase chain reaction determined the expression levels of CHOP, BIM, and BID. Immunostaining with microtubule-associated protein 1 light chain 3 measured autophagy activity. Trypan blue exclusion determined the cell viability. Results TM treatment increased the messenger RNA levels of CHOP and BIM but decreased the messenger RNA levels of BID. TM increased autophagy and decreased cell viability. Individual inhibition of CHOP, BIM, or BID protected against autophagy and cell death. However, simultaneous treatment with any combination of CHOP, BIM, and BID small interfering RNAs reduced autophagy activity but increased cell death independent of ER stress induction. Conclusions Autophagy in hepatocytes results from acute ER stress and involves interplay, at the gene expression level, of CHOP, BIM, and BID. Inhibition of any one of these individual genes during acute ER stress is protective against cell death. Conversely, inhibition of any two of the three genes results in increased nonautophagic cell death independent of ER stress induction. This study suggests interplay between CHOP, BIM, and BID expression that can be leveraged for protection against ER stress-related cell death. However, disruption of the CHOP/BH3 gene expression homeostasis is detrimental to cell survival independent of other cellular stress.

Apoptosis plays an important role in the mechanism regulating the development of glomerulonephritis. We investigated whether polymorphisms of apoptotic genes such as B-cell CLL/lymphoma 2 (BCL2), BH3-interacting domain death agonist (BID), and caspase 8 (CASP8) were associated with immunoglobulin A nephropathy (IgAN) and with the clinical phenotypes of IgAN patients.We genotyped promoter and coding region single nucleotide polymorphisms (SNPs) (rs2279115 and rs1801018 for BCL2; rs8190315 and rs2072392 for BID; and rs6747918 and rs1045487 for CASP8) using direct sequencing in 195 IgAN patients and 289 control subjects.No SNPs were associated with IgAN. However, in analysis of clinical phenotypes, we found that rs8190315 and rs2072392 of BID were associated with proteinuria levels of IgAN patients in additive (AG vs. GG vs. AA, p = 0.0008 for rs8190315; TC vs. CC vs. TT, p = 0.0012 for rs2072392) and dominant models (AG/GG vs. AA, p = 0.0014 for rs8190315; TC/CC vs. TT, p = 0.0031 for rs2072392). In particular, the frequencies of genotypes containing minor alleles of rs8190315 (G allele) and rs2072392 (C allele) were increased in IgAN patients with higher protienuria levels (40 mg/m(2)/h).These results suggest that BID may play a role in severe IgAN.

TNF-α transduces signals of survival or death via its two receptors, R1/p55/p60 and RII/p80/p75. The role of caspases as effectors of cell death is universally accepted, although caspase inhibitors may potentiate TNF cytotoxicity in some instances. In conditions when macromolecular synthesis is blocked, caspases are part of the machinery that executes TNF-triggered apoptotic death in U937, a human myelomonocyte cell line, and in the Jurkat T cell line. However, inhibition of p38 mitogen-activated protein kinase (p38 MAPK) triggered TNF cytotoxicity in U937 cells and murine splenic macrophages, but not the Jurkat cell line. TNF induced expression of the antiapoptotic protein c-IAP2 (cytoplasmic inhibitor of apoptosis protein 2), and was blocked in the presence of a p38 MAPK inhibitor, which also induced caspase-dependent, TNF-mediated apoptosis in U937 cells. Thus, inhibition of p38 MAPK resulted in the activation of caspase 9 and cleavage of the adaptor molecule BH3 interacting domain death agonist, and blocked NF-κB-mediated transactivation, without affecting the nuclear translocation of NF-κB. Collectively, these data show that activation of p38 MAPK is critical to cell survival by TNF in U937 cells, and demonstrate lineage-specific regulation of TNF-triggered signals of activation or apoptosis.

We report here the purification of a cytosolic protein that induces cytochrome c release from mitochondria in response to caspase-8, the apical caspase activated by cell surface death receptors such as Fas and TNF. Peptide mass fingerprinting identified this protein as Bid, a BH3 domain–containing protein known to interact with both Bcl2 and Bax. Caspase-8 cleaves Bid, and the COOH-terminal part translocates to mitochondria where it triggers cytochrome c release. Immunodepletion of Bid from cell extracts eliminated the cytochrome c releasing activity. The cytochrome c releasing activity of Bid was antagonized by Bcl2. A mutation at the BH3 domain diminished its cytochrome c releasing activity. Bid, therefore, relays an apoptotic signal from the cell surface to mitochondria.

We report here that BID, a BH3 domain–containing proapoptotic Bcl2 family member, is a specific proximal substrate of Casp8 in the Fas apoptotic signaling pathway. While full-length BID is localized in cytosol, truncated BID (tBID) translocates to mitochondria and thus transduces apoptotic signals from cytoplasmic membrane to mitochondria. tBID induces first the clustering of mitochondria around the nuclei and release of cytochrome c independent of caspase activity, and then the loss of mitochondrial membrane potential, cell shrinkage, and nuclear condensation in a caspase-dependent fashion. Coexpression of Bclx L inhibits all the apoptotic changes induced by tBID. Our results indicate that BID is a mediator of mitochondrial damage induced by Casp8.

The tumor suppressor p53 regulates the expression of genes involved in cell cycle progression, senescence and apoptosis. Here, we investigated the effect of single point mutations in the oligomerization domain (OD) on tetramerization, transcription, ubiquitylation and stability of p53. As predicted by docking and molecular dynamics simulations, p53 OD mutants show functional defects on transcription, Mdm2-dependent ubiquitylation and 26S proteasome-mediated degradation. However, mutants unable to form tetramers are well degraded by the 20S proteasome. Unexpectedly, despite the lower structural stability compared to WT p53, p53 OD mutants form heterotetramers with WT p53 when expressed transiently or stably in cells wild type or null for p53. In consequence, p53 OD mutants interfere with the capacity of WT p53 tetramers to be properly ubiquitylated and result in changes of p53-dependent protein expression patterns, including the pro-apoptotic proteins Bax and PUMA under basal and adriamycin-induced conditions. Importantly, the patient derived p53 OD mutant L330R (OD1) showed the more severe changes in p53-dependent gene expression. Thus, in addition to the well-known effects on p53 stability, ubiquitylation defects promote changes in p53-dependent gene expression with implications on some of its functions.