Necroptosis Inhibitor Necrostatin-1 Research Articles

RIP3 induces ischemic neuronal DNA degradation and programmed necrosis in rat via AIF.

We have reported that nuclear translocation of Receptor-interacting protein 3 (RIP3) involves in neuronal programmed necrosis after 20-min global cerebral ischemia/reperfusion (I/R) injury. Herein, the underlying mechanisms and the nuclear role of RIP3 were investigated further. The necroptosis inhibitor necrostatin-1 (Nec-1), the autophagy inhibitor 3-methyladenine (3-MA), and the caspase-3 inhibitor acetyl-L-aspartyl-L-methionyl-L-glutaminyl-L-aspart-1-al (Ac-DMQD-CHO) were administered intracerebroventricularly 1 h before ischemia. Protein expression, location and interaction was determined by western blot, immunofluorescence or immunoprecipitation. Most CA1 neuronal death induced by 20-min global cerebral I/R injury was TUNEL-positive. Neuronal death and rat mortality rates were greatly inhibited by Nec-1 and 3-MA pre-treatment, but not by Ac-DMQD-CHO. And no activation of caspase-3 was detected after I/R injury. Caspase-8 was expressed richly in GFAP-positive astrocytes and Iba-1-positive microglia, but was not detected in Neun-positive neurons. The nuclear translocation and co-localization of RIP3 and AIF, and their interaction were detected after I/R injury. These processes were inhibited by Nec-1 and 3-MA pre-treatment, but not by Ac-DMQD-CHO. The formation of an RIP3-AIF complex and its nuclear translocation are critical to ischemic neuronal DNA degradation and programmed necrosis. Neurons are more likely to enter the programmed necrosis signal pathway for the loss of caspase-8 suppression.

Activation of innate antiviral immune response via double-stranded RNA-dependent RLR receptor-mediated necroptosis.

Viruses induce double-stranded RNA (dsRNA) in the host cells. The mammalian system has developed dsRNA-dependent recognition receptors such as RLRs that recognize the long stretches of dsRNA as PAMPs to activate interferon-mediated antiviral pathways and apoptosis in severe infection. Here we report an efficient antiviral immune response through dsRNA-dependent RLR receptor-mediated necroptosis against infections from different classes of viruses. We demonstrated that virus-infected A549 cells were efficiently killed in the presence of a chimeric RLR receptor, dsCARE. It measurably suppressed the interferon antiviral pathway but promoted IL-1β production. Canonical cell death analysis by morphologic assessment, phosphatidylserine exposure, caspase cleavage and chemical inhibition excluded the involvement of apoptosis and consistently suggested RLR receptor-mediated necroptosis as the underlying mechanism of infected cell death. The necroptotic pathway was augmented by the formation of RIP1-RIP3 necrosome, recruitment of MLKL protein and the activation of cathepsin D. Contributing roles of RIP1 and RIP3 were confirmed by gene knockdown. Furthermore, the necroptosis inhibitor necrostatin-1 but not the pan-caspase inhibitor zVAD impeded dsCARE-dependent infected cell death. Our data provides compelling evidence that the chimeric RLR receptor shifts the common interferon antiviral responses of infected cells to necroptosis and leads to rapid death of the virus-infected cells. This mechanism could be targeted as an efficient antiviral strategy.

Cigarette smoke-induced necroptosis and DAMP release trigger neutrophilic airway inflammation in mice.

Recent data indicate a role for airway epithelial necroptosis, a regulated form of necrosis, and the associated release of damage-associated molecular patterns (DAMPs) in the development of chronic obstructive pulmonary disease (COPD). DAMPs can activate pattern recognition receptors (PRRs), triggering innate immune responses. We hypothesized that cigarette smoke (CS)-induced epithelial necroptosis and DAMP release initiate airway inflammation in COPD. Human bronchial epithelial BEAS-2B cells were exposed to cigarette smoke extract (CSE), and necrotic cell death (membrane integrity by propidium iodide staining) and DAMP release (i.e., double-stranded DNA, high-mobility group box 1, heat shock protein 70, mitochondrial DNA, ATP) were analyzed. Subsequently, BEAS-2B cells were exposed to DAMP-containing supernatant of CS-induced necrotic cells, and the release of proinflammatory mediators [C-X-C motif ligand 8 (CXCL-8), IL-6] was evaluated. Furthermore, mice were exposed to CS in the presence and absence of the necroptosis inhibitor necrostatin-1, and levels of DAMPs and inflammatory cell numbers were determined in bronchoalveolar lavage fluid. CSE induced a significant increase in the percentage of necrotic cells and DAMP release in BEAS-2B cells. Stimulation of BEAS-2B cells with supernatant of CS-induced necrotic cells induced a significant increase in the release of CXCL8 and IL-6, in a myeloid differentiation primary response gene 88-dependent fashion. In mice, exposure of CS increased the levels of DAMPs and numbers of neutrophils in bronchoalveolar lavage fluid, which was statistically reduced upon treatment with necrostatin-1. Together, we showed that CS exposure induces necrosis of bronchial epithelial cells and subsequent DAMP release in vitro, inducing the production of proinflammatory cytokines. In vivo, CS exposure induces neutrophilic airway inflammation that is sensitive to necroptosis inhibition.

Abstract 2700: The IAP inhibitor Debio 1143 reverses carboplatin resistance in ovarian cancer cells by inducing both apoptosis and necroptosis

Abstract Ovarian cancer remains the most lethal gynecological cancer. The efficacy of conventional chemotherapy with a combination of platinum salts and taxanes is transient because 70 to 80% of patients initially responding to platinum finally relapse. Mechanisms of resistance to carboplatin include a disruption of the balance between key pro-and anti-apoptotic proteins such as the inhibitor of apoptosis protein (IAP) family. Drug mimetics of the endogenous IAP inhibitor SMAC constitute a novel therapeutic approach to overcome apoptosis resistance and are currently in clinical development for cancer treatment. In this study we tested the ability of the oral IAP inhibitor Debio 1143 to revert carboplatin resistance in three carboplatin-sensitive (OVCAR-3, IGROV-1, A2780S) and three carboplatin-resistant (SKOV-3, A2780R, EFO-21) human ovarian tumor cell lines. We demonstrated that in vitro Debio 1143 was able to sensitize SKOV-3, EFO-21 and A2780R ovarian tumor cells to carboplatin. In addition, we demonstrated that the combination allowed a full recovery of carboplatin sensitivity in A2780R similar to the sensitive cell line A2780S. The sensitization to carboplatin correlated with an increased induction of apoptosis (Annexin V/PI assay and PARP cleavage) in SKOV-3 and A2780R cells. In EFO-21 cells, Debio 1143 triggered necroptosis as shown by the necroptosis inhibitor necrostatin-1 which was able to suppress Debio 1143-induced cell death with or without carboplatin. Our study shows that in the above human ovarian tumor cell lines Debio 1143 induces degradation of cIAP1 while XIAP levels remain unchanged. Using a carboplatin-resistant in vivo ovarian tumor mouse model, we further showed that Debio 1143 alone or in combination with carboplatin causes marked regression of subcutaneous SKOV-3 tumors. Together, our results suggest that Debio 1143 in combination with carboplatin could provide considerable benefit to patients with innate or acquired resistance to carboplatin by acting as a sensitizing agent. Debio 1143 is currently evaluated in combination with carboplatin and paclitaxel in patient with advanced solid malignancies including platinum-refractory ovarian cancer (NCT01930292). Citation Format: Benoît Thibault, Ludivine Genre, Clothilde Broca, Maryse Barbier, Claudio Zanna, Grégoire Vuagniaux, Jean-Pierre Delord, Bettina Couderc. The IAP inhibitor Debio 1143 reverses carboplatin resistance in ovarian cancer cells by inducing both apoptosis and necroptosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2700. doi:10.1158/1538-7445.AM2015-2700

Abstract 17348: Necrostatin 7 Limits Myocardial Infarct Size and Reduces Cardiac Remodeling After Permanent Coronary Occlusion in Rats

Background: Reduction of irreversible myocardial ischemia-reperfusion injury (IRI) remains important. One of the promising strategies aimed at myocardial IRI alleviation is modulation of programmed cell death (PCD) pathways. PCD mode displaying morphological characteristics of necrosis, and amenable to pharmacological manipulation is referred to as necroptosis. Necroptosis inhibitor necrostatin-1 has been shown to exert cardio- and neuroprotective effects. In the present work, the effect of necrostatin-7 (Nec-7) on myocardial injury in the rat model of permanent coronary occlusion was studied. Methods: Male Wistar rats (n = 19) were anesthetized with pentobarbital. The animals were subjected to permanent coronary occlusion (PCO) and intraperitoneal (i.p.) Nec-7 administration 1 h prior to PCO at a dose of 14.5 mg/kg in dimethyl sulfoxide (DMSO) or DMSO alone at a dose of 3.1 g/kg. Control rats were treated with saline. Three weeks after PCO, serum levels of NT-proBNP were measured, and histological outcomes were assessed. The infarct size (IS, %) and infarct length (IL, mm) were analyzed morphometrically. Results: DMSO caused significant reduction in serum NT-proBNP level vs. Control (0.3 ± 0.19 vs. 0.5 ± 0.22 ng/ml, p = 0.001), while Nec-7 further decreased NT-proBNP level in comparison with DMSO (0.2 ± 0.14 ng/ml, p = 0.008 vs. DMSO). Compared with Control, DMSO reduced adverse left ventricular remodeling, as evidenced by reduction in IS (16.0 ± 2.92 and 12.9 ± 1.72%, p = 0.015) and IL (6.2 ± 0.89 and 3.8 ± 0.35 mm, p = 0.008). Nec-7 treatment resulted in additional reduction of both IS and IL vs. DMSO group (9.0 ± 4.91 % and 2.9 ± 1.62 mm, respectively; p = 0.013 and p = 0.011 vs. DMSO, respectively). Conclusion: Nec-7 has cardioprotective properties, reducing myocardial wall stress and myocardial remodeling in the rat model of myocardial infarction.

Necroptosis is associated with low procaspase-8 and active RIPK1 and -3 in human glioma cells.

Necroptosis is a regulated necrotic cell death that involves receptor-interacting protein kinases RIPK1 and RIPK3. Here, we report that edelfosine triggers a rapid and massive cell death in human glioblastoma cells with characteristics of necrosis. Only a minor proportion of edelfosine-treated cells underwent caspase-dependent apoptosis. Autophagy and a rapid influx of extracellular calcium into the cells had little impact on cell death. Levels of procaspase-8 were very low in necroptosis-prone glioma cells compared with the levels in other cancer cell types that underwent apoptosis upon edelfosine treatment. The RIPK1-dependent necroptosis inhibitors necrostatin-1 (Nec-1) and Nec-1s as well as siRNA-mediated silencing of RIPK3 inhibited edelfosine-induced necroptosis, resulting in increased caspase-dependent apoptosis in edelfosine-treated glioblastoma U118 cells. Inhibition of the RIPK3 substrate MLKL with necrosulfonamide also increased apoptosis in edelfosine-treated cells. These data support a major role for RIPK1 and RIPK3 in the induction of necrotic cell death and in the switch from necrosis to apoptosis following edelfosine treatment. These results indicate that the ether lipid edelfosine exerts a rapid necroptotic cell death in apoptosis-reluctant glioblastoma cells, suggesting that induction of necroptosis could constitute a new approach for glioblastoma therapy.

BIM upregulation and ROS-dependent necroptosis mediate the antitumor effects of the HDACi Givinostat and Sorafenib in Hodgkin lymphoma cell line xenografts

Relapsed/refractory Hodgkin's lymphoma (HL) is an unmet medical need requiring new therapeutic options. Interactions between the histone deacetylase inhibitor Givinostat and the RAF/MEK/ERK inhibitor Sorafenib were examined in HDLM-2 and L-540 HL cell lines. Exposure to Givinostat/Sorafenib induced a synergistic inhibition of cell growth (range, 70-80%) and a marked increase in cell death (up to 96%) due to increased H3 and H4 acetylation and strong mitochondrial injury. Gene expression profiling indicated that the synergistic effects of Givinostat/Sorafenib treatment are associated with the modulation of cell cycle and cell death pathways. Exposure to Givinostat/Sorafenib resulted in sustained production of reactive oxygen species (ROS) and activation of necroptotic cell death. The necroptosis inhibitor Necrostatin-1 prevented Givinostat/Sorafenib-induced ROS production, mitochondrial injury, activation of BH3-only protein BIM and cell death. Knockdown experiments identified BIM as a key signaling molecule that mediates Givinostat/Sorafenib-induced oxidative death of HL cells. Furthermore, in vivo xenograft studies demonstrated a 50% reduction in tumor burden (P<0.0001), a 5- to 15-fold increase in BIM expression (P < 0.0001) and a fourfold increase in tumor necrosis in Givinostat/Sorafenib-treated animals compared with mice that received single agents. These results provide a rationale for exploring Givinostat/Sorafenib combination in relapsed/refractory HL.

Necroptosis inhibitor necrostatin-1 promotes cell protection and physiological function in traumatic spinal cord injury

Spinal cord injury (SCI) is a common and serious trauma which lacks efficient treatment. Inhibition of cell death in the trauma area is important for spinal cord protection during this process. In this study, necroptosis inhibitor necrostatin-1 (Nec-1) was used to treat SCI rats, to investigate the role of Nec-1 in the recovery of SCI. Nec-1 was found to reduce lesions, cytokines and reactive oxygen species (ROS), improve pathological conditions and blood supply in the spinal cord trauma area. Further study indicated that Nec-1 could inhibit necroptosis by inhibiting RIP1/3–MLKL recruitment and inhibit apoptosis by inhibiting Caspase 3 and Bax while activating Bcl-2. Ethological performance of SCI rats confirmed improvement and protection of physiological function by Nec-1. Nec-1 as a potential treatment for SCI warrants further study. To our knowledge, this is the first study on the role of Nec-1 in the treatment of traumatic SCI. Our research also found inhibition effects of Nec-1 on apoptosis, not only necroptosis – as reported by most publications.

The anti-tumor effect of shikonin on osteosarcoma by inducing RIP1 and RIP3 dependent necroptosis

BackgroundOsteosarcoma is the most frequent primary malignant bone tumor, notorious for its lung metastasis. Shikonin, an effective constituent extracted from Chinese medicinal herb, was demonstrated to induce necroptosis in some cancers.MethodsMTT assay was performed to detect cell survival rate in vitro. Flow cytometry was used to analyze cell cycle and cell death. Western blot was performed to determine the expression levels of RIP1, RIP3, caspase-3, caspase-6 and PARP. The tibial primary and lung metastatic osteosarcoma models were used to evaluate the anti-tumor effect of shikonin in vivo.ResultsThe cell survival rate was decreased in a dose and time dependent manner when treated with shikonin. No major change in cell cycle was observed after shikonin treatment. The cell death induced by shikonin could be mostly rescued by specific necroptosis inhibitor necrostatin-1, but not by general caspase inhibitor Z-VAD-FMK. The number of necrotic cells caused by shikonin was decreased after being pretreated with Nec-1 detected by flow cytometry in K7 cells. After 8-hour treatment of shikonin, the expression levels of RIP1 and RIP3 were increased while caspase-3, caspase-6 and PARP were not activated in K7 and U2OS cells determined by Western blot. Size of primary tumor and lung metastasis in shikonin treated group were significantly reduced. The protein levels of RIP1 and RIP3 in primary tumor tissues were increased by shikonin. The overall survival of lung metastatic models was longer compared with control group (p < 0.001).ConclusionsShikonin had prompt but profound anti-tumor effect on both primary and metastatic osteosarcoma, probably by inducing RIP1 and RIP3 dependent necroptosis. Shikonin would be a potential anti-tumor agent on the treatment of primary and metastatic osteosarcoma.

Neoalbaconol induces energy depletion and multiple cell death in cancer cells by targeting PDK1-PI3-K/Akt signaling pathway

Many natural compounds derived from plants or microbes show promising potential for anticancer treatment, but few have been found to target energy-relevant regulators. In this study, we report that neoalbaconol (NA), a novel small-molecular compound isolated from the fungus, Albatrellus confluens, could target 3-phosphoinositide-dependent protein kinase 1 (PDK1) and inhibit its downstream phosphoinositide-3 kinase (PI3-K)/Akt-hexokinase 2 (HK2) pathway, which eventually resulted in energy depletion. By targeting PDK1, NA reduced the consumption of glucose and ATP generation, activated autophagy and caused apoptotic and necroptotic death of cancer cells through independent pathway. Necroptosis was remarkably induced, which was confirmed by several necroptosis-specific markers: the activation of autophagy, presence of necrotic morphology, increase of receptor-interacting protein 1 (RIP1)/RIP3 colocalization and interaction and rescued by necroptosis inhibitor necrostatin-1. The possibility that Akt overexpression reversed the NA-induced energy crisis confirmed the importance of the PDK1-Akt-energy pathway in NA-mediated cell death. Moreover, NA shows the capability to inhibit PI3-K/Akt signaling and suppress tumor growth in the nasopharyngeal carcinoma (NPC) nude mouse model. These results supported the feasibility of NA in anticancer treatments.

Necrostatin-5 limits infarct size in the isolated rat heart

Background: Inhibition of ischemia-reperfusion-induced programmed cardiac myocyte death has been considered as a promising therapeutic strategy for myocardial infarction. Necroptosis represents one of the recently discovered types of programmed cell death which could be manipulated by the pharmacological inhibitors – necrostatins. The aim of the present study was to study the effect of necroptosis inhibitors necrostatin-1 and -5 (Nec-1 and Nec-5, respectively) on myocardial infarct size in the isolated rat heart subjected to global ischemia-reperfusion. Methods: Male Wistar rats were anesthetized with pentobarbital sodium. The isolated Langendorff-perfused hearts were subjected to 30-min global ischemia and 120-min reperfusion. Infarct size was determined histochemically with triphenyltetrazolium chloride staining. Left ventricular pressures, heart rate, and coronary flow were monitored throughout the experiments. Nec-1 and -5 were dissolved in dimethyl sulfoxide (DMSO). The hearts were randomized into one of six groups: 1) controls (n = 8); 2) DMSO (n = 8) – the animals received intraperitoneal (i.p.) injection of vehicle 60 min prior to heart isolation; 3) intracoronary (i.c.) DMSO (n = 5) – the hearts were perfused with 64.7 mmol/L DMSO for 6.5 minutes before ischemia; 4) Nec-1 i.p. (n = 8) – Nec-1 was administered i.p. 60 min prior to heart isolation at a dose of 1.65 mg/kg; 5) Nec-5 i.p. (n = 13) – Nec-5 was administered in the same way as in the previous group at a dose of 2.46 mg/kg; 6) Nec-1 i.c. (n = 6) - the hearts were perfused with 44.5 mmol/L Nec-1 for 6.5 minutes before ischemia. Results: I.p. administration of DMSO caused a significant reduction in infarct size compared with controls (35.0±2.05 vs. 50.0±3.90%, respectively, p = 0.0042). I.p. injection of Nec-1 (20.9±3.02%) and Nec-5 (16.6±1.82%) further decreased infarct size in comparison with i.p. DMSO group (p = 0.0077 and 0.00048, respectively). I.c. administration of Nec-1 resulted in significant increase in the left ventricular systolic pressure (by 26.8±2.77% from baseline value). The positive inotropic effect of Nec-1 was limited to the period of drug administration. Nec-1 failed to reduce infarct size after preischemic i.c. administration (40.2±5.71%, vs. 50.8±2.11% in i.c. DMSO group, p = 0.175). Conclusion: This is the first study demonstrating the infarct-limiting effect of Nec-5. In addition, the positive inotropic effect of i.c. Nec-1 is described. Our data corroborate previous findings on the presence on infarct-limiting effect of Nec-1 in the setting of myocardial ischemia-reperfusion.

Study of Cardioprotective Effects of Necroptosis Inhibitors on Isolated Rat Heart Subjected to Global Ischemia–Reperfusion

The cardioprotective effects of necroptosis inhibitors necrostatin-1 and necrostatin-5 were studied on the isolated heart model in rats. Intraperitoneal injection of necrostatin-1 (1.65 mg/kg) or necrostatin-5 (2.46 mg/kg) 60 min before reperfusion of the isolated heart reduced the infarction zone caused by 30-min global ischemia and 120-min reperfusion. Intracoronary injection of necrostatin-1 (44.5 μmol/liter) caused an increase of left-ventricular systolic pressure, that is produced a positive inotropic effect, but did not reduce the infarction zone.

The Inhibition of ERK Activation Mediates the Protection of Necrostatin-1 on Glutamate Toxicity in HT-22 Cells

Receptor-interacting protein 1 (RIP1), a molecular switch protein from apoptosis to necroptosis, is regarded to play an essential role in necroptotic cell death. Although the increased RIP1 activity induced by tumor necrosis factor α activates mitogen-activated protein kinases (MAPKs) including ERK and leads to apoptotic or necrotic cell death, it is unclear what is the role of ERK during the process of necroptosis. In this study, our data demonstrated that ERK inhibitors U0126 and PD98059 blocked glutamate-induced necroptosis in HT-22 cells, indicating the critical role of ERK activation in necroptosis. Further, we found glutamate treatment increased phosphorylated ERK1/2 level, but the specific necroptosis inhibitor Necrostatin-1 (Nec-1) significantly inhibited the phosphorylation of ERK1 (P44) at 5, 10, and 15min after glutamate treatment; the phosphorylation of ERK2 (P42) level was also markedly reduced by Nec-1 at 10min after glutamate treatment. The phosphorylation of JNK and P38, two other MAPK members, were slightly increased after glutamate treatment, but Nec-1 had no inhibitory effect on JNK and P38 activation. Our finding suggested that ERK activation may play an important role in necroptotic cell death and the inhibition of ERK activation mediated the protection of Nec-1 on glutamate-induced necroptosis. Since ERK is considered as a downstream of RIP1, the RIP1/ERK signal pathway may provide new therapeutic avenues for the treatment of ischemia-reperfusion damage and neurodegenerative diseases-containing necroptotic cell death.

Sulphur Antioxidants Inhibit Oxidative Stress Induced Retinal Ganglion Cell Death by Scavenging Reactive Oxygen Species but Influence Nuclear Factor (Erythroid-Derived 2)-Like 2 Signalling Pathway Differently

This study aimed to show if two different sulphur containing drugs sulbutiamine and acetylcysteine (NAC) could attenuate the effects of two different insults being serum deprivation and glutamate/buthionine sulfoximine (GB)-induced death to transformed retinal ganglion cell line (RGC-5) in culture. Cells were exposed to either 5 mM of GB for 24 h or serum deprivation for 48 h with inclusion of either NAC or sulbutiamine. Cell viability, microscopic evidence for apoptosis, caspase 3 activity, reactive oxygen species (ROS), glutathione (GSH), catalase and gluthathione-S-transferase (GST) were determined. The effects of NAC and sulbutiamine on the oxidative stress related transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) levels and its dependent phase II enzyme haemeoxygenase-1 (HO-1) were carried out using Western blot and quantitative-polymerase chain reaction (PCR). NAC and sulbutiamine dose-dependently attenuated serum deprivation-induced cell death. However NAC but not sulbutiamine attenuated GB-induced cell death. NAC and sulbutiamine both independently stimulated the GSH and GST production but scavenged different types of ROS with different efficacy. Moreover only sulbutiamine stimulated catalase and significantly increased Nrf-2 and HO-1 levels. In addition, the pan caspase inhibitor, benzoylcarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk) attenuated the negative effect of serum deprivation while the necroptosis inhibitor (necrostatin-1) counteracted solely an insult of GB. The neuroprotective actions of NAC and sulbutiamine in GB or serum-deprivation insult are therefore different.

Abstract 2277: Autophagy-associated necroptosis contributes to cancer cell cytotoxicity induced by the chalcone compound SBC2

Abstract Chalcone-based compounds are potential agents for cancer chemoprevention and chemotherapy. The recently developed chalcone-based compound SBC2 suppresses xenograft tumor growth in nude mice, however, the mechanisms by which SBC2 kills cancer cells are elusive. In this study, we have investigated the cellular signaling pathways involved in SBC2's cytotoxicity in cancer cells derived from lung and bladder tumors. SBC2 potently killed different cancer cells, which was effectively blocked by the necroptosis inhibitor Necrostatin-1 and knockdown of the necroptosis pathway components RIP1 and RIP3, suggesting that SBC2-induced cytotoxicity is associated with necroptosis. SBC2 robustly activated JNK and ERK through activation of their upstream activating kinases MEKK1 and MKK7. Blocking JNK and ERK effectively blocked SBC2-induced cytotoxicity, implying an important role of these MAPKs in SBC2-induced cancer cell death. In addition, SBC2 strongly induced autophagy, which was suppressed when JNK was blocked. Furthermore, SBC2 triggered BCL-XL phosphorylation that was dependent on JNK. Importantly, suppression of autophagy with either pharmacological inhibitors or a siRNA targeting the essential autophagy component ATG7 effectively attenuated SBC2-induced cell death. These results suggest that SBC2 kills cancer cells through activating JNK-mediated and autophagy-associated necroptosis. Taken together, our observations substantiate SBC2 as a potential anticancer therapeutic and identify a novel pathway for cancer cell killing by SBC2 that involves autophagy and necroptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2277. doi:1538-7445.AM2012-2277

Cyclic AMP induces necroptotic cell death in cardiac fibroblasts

Increased levels of the second messenger cyclic AMP (cAMP) can promote cell loss of a variety of cell types. The precise mechanism(s) for this loss is/are not clearly defined, although cAMP can promote apoptosis of certain types of cells (Insel et al, Acta Physiol in press). In the present study, we used adult rat cardiac fibroblasts (rCFs) to test the hypothesis that cAMP induces necroptosis. Our results from morphological and biochemical studies provide evidence that cAMP induces necroptosis in rCFs and is mediated by protein kinase A and not Epac, another cAMP effector. The findings include destruction of the plasma membrane of rCFs, which differs from apoptotic shrinking of the membrane, and a prominent increase in expression of HMGB1, a marker of necrosis, following treatment with cAMP‐elevating agents. In addition, such agents induce death in a caspase‐PARP independent manner. The pan‐caspase inhibitor, z‐VAD‐fmk increases cell death induced by CPT‐cAMP but the necroptosis inhibitors necrostatin‐1 and cyclosporine A reduce this effect. Taken together, these findings lead us to conclude that cAMP induces necroptosis in rCFs and that this occurs via a PKA‐mediated mechanism. cAMP/PKA‐promoted necroptosis may contribute to decrease in number of CFs in vivo and may be a mechanism of cAMP‐promoted cell death in other types of cells. (Supported by research and training grants from NIH).

Reactive oxygen species‐dependent necroptosis in Jurkat T cells induced by pathogenic free‐living Naegleria fowleri

Naegleria fowleri, a free-living amoeba, is the causative pathogen of primary amoebic meningoencephalitis in humans and experimental mice. N.fowleri is capable of destroying tissues and host cells through lytic necrosis. However, the mechanism by which N.fowleri induces host cell death is unknown. Electron microscopy indicated that incubation of Jurkat T cells with N.fowleri trophozoites induced necrotic morphology of the Jurkat T cells. N.fowleri also induced cytoskeletal protein cleavage, extensive poly (ADP-ribose) polymerase hydrolysis and lactate dehydrogenase (LDH) release. Although no activation of caspase-3 was observed in Jurkat T cells co-incubated with amoebae, intracellular reactive oxygen species (ROS) were strongly generated by NADPH oxidase (NOX). Pretreating cells with necroptosis inhibitor necrostatin-1 or NOX inhibitor diphenyleneiodonium chloride (DPI) strongly inhibited amoeba-induced ROS generation and Jurkat cell death, whereas pan-caspase inhibitor z-VAD-fmk did not. N.fowleri-derived secretory products (NfSP) strongly induced intracellular ROS generation and cell death. Necroptotic effects of NfSP were effectively inhibited by pretreating NfSP with proteinase K. Moreover, NfSP-induced LDH release and intracellular ROS accumulation were inhibited by pretreating Jurkat T cells with DPI or necrostatin-1. These results suggest that N.fowleri induces ROS-dependent necroptosis in Jurkat T cells.

TNFα-Induced Necroptosis and Autophagy via Supression of the p38–NF-κB Survival Pathway in L929 Cells

Tumor necrosis factor alpha (TNFα) has been reported to induce necroptosis and autophagy, but its mechanisms remain unclear. In this study, we found that TNFα significantly induced necroptosis and autophagy in murine fibrosarcoma L929 cells. The necroptosis inhibitor necrostatin-1 (Nec-1) completely blocked TNFα-induced necroptosis and autophagy, but inhibition of autophagy with 3-methyladenine (3MA) or Beclin 1 small interfering RNA (siRNA) promoted necroptosis, indicating that autophagy acted as a negative regulator of TNFα-induced necroptosis. The cytotoxicity of TNFα was accompanied by decreased expressions of phosphorylated p38 mitogen-activated protein kinase (p-p38) and nuclear factor-kappa B (NF-κB), and inhibition of p38 and NF-κB activation by chemical inhibitors or siRNA augmented these necroptotic and autophagic responses to TNFα in the cells. The pan-caspase inhibitor z-VAD-fmk (zVAD) exacerbated TNFα-induced necroptosis and autophagy. Combined treatment with TNFα and zVAD further decreased the expressions of p-p38 and NF-κB compared with TNFα alone treatment. Consequently, these results indicated that suppression of the p38-NF-κB survivial signaling pathway promoted necroptotic and autophagic cell death in TNFα-treated L929 cells.

Synergistic protective effects of humanin and necrostatin-1 on hypoxia and ischemia/reperfusion injury

Since several different pathways are involved in cerebral ischemia/reperfusion injury, combination therapy rather than monotherapy may be required for efficient neuroprotection. In this study, we examined the protective effects of an apoptosis inhibitor Gly 14-humanin (HNG) and a necroptosis inhibitor necrostatin-1 (Nec-1) on hypoxia/ischemia/reperfusion injury. Cultured mouse primary cortical neurons were incubated with Nec-1, HNG or both in a hypoxia chamber for 60 min. Cell viability was determined by MTS assay at 24 h after oxygen-glucose deprivation (OGD) treatment. Mice underwent middle cerebral artery occlusion for 75 min followed by 24 h reperfusion. Mice were administered HNG and/or Nec-1 (i.c.v.) at 4 h after reperfusion. Neurological deficits were evaluated and the cerebral infarct volume was determined by TTC staining. Nec-1 or HNG alone had protective effects on OGD-induced cell death. Combined treatment with Nec-1 and HNG resulted in more neuroprotection than Nec-1 or HNG alone. Treatment with HNG or Nec-1 reduced cerebral infarct volume from 59.3 ± 2.6% to 47.0 ± 2.3% and 47.1 ± 1.5%, respectively. Combined treatment with HNG and Nec-1 improved neurological scores and decreased infarct volume to 38.6 ± 1.5%. In summary, we demonstrated that the combination treatment of HNG and Nec-1 conferred synergistic neuroprotection on hypoxia/ischemia/reperfusion injury in vitro and in vivo. These findings provide a novel therapeutic strategy for the treatment of stroke by combining anti-apoptosis and anti-necroptosis therapy.

Apoptosis and necroptosis are induced in rainbow trout cell lines exposed to cadmium

Cadmium is an important environmental toxicant that can kill cells. A number of studies have implicated apoptosis as well as necrosis and, most recently, a form of programmed necrosis termed necroptosis in the process of cadmium-mediated toxicity, but the exact mechanism remains ill-defined and may depend on the affected cell type. This study investigated which mode of cell death may be responsible for cell death induction in cadmium-exposed trout cell lines from gill and liver and if this cell death was sensitive to inhibitors of necroptosis or apoptosis, respectively. It was observed that intermediate levels of cadmium that killed approximately 50% of the cells over 96–120 h of exposure caused cell death that morphologically resembled apoptosis and was associated with an increase of apoptotic markers such as the number of cells with diminished DNA content (sub-G1 cells), condensed or fragmented nuclei, and elevation of caspase-3 activity. At the same time, however, cells also lost plasma membrane integrity, as indicated by uptake of propidium iodide, showed a decrease of ATP levels and mitochondrial membrane potential, and displayed cell swelling, signs associated with secondary necrosis, or equally possible, necroptotic cell death. Importantly, many of these alterations were at least partly inhibited by the necroptosis inhibitor necrostatin-1 and were to a lesser extent also sensitive to the pan-caspase inhibitor zVAD-fmk, indicating that multiple modes of cell death are concurrently induced in cadmium-exposed trout cells, including necroptosis and apoptosis. Cell death appeared to lack concurrent radical formation, consistent with genetically regulated necroptotic cell death, but was characterized by the rapid induction of DNA damage markers, and the early onset of disintegration of the Golgi complex. Comparative experiments evaluating copper-toxicity indicated that in comparison to cadmium much higher concentrations of this metal were required to induce cell death and that neither necrostatin-1 nor a pan-caspase inhibitor conferred protection, suggesting that additional modes of cell death can be triggered in response to poisoning with heavy metals.