Caspase Inhibitor Z-VAD Research Articles

Pyroptosis leads to loss of centrosomal integrity in macrophages

NLRP3 forms a multiprotein inflammasome complex to initiate the inflammatory response when macrophages sense infection or tissue damage, which leads to caspase-1 activation, maturation and release of the inflammatory cytokines interleukin-1β (IL-1β) and IL-18 and Gasdermin-D (GSDMD) mediated pyroptosis. NLRP3 inflammasome activity must be controlled as unregulated and chronic inflammation underlies inflammatory and autoimmune diseases. Several findings uncovered that NLRP3 inflammasome activity is under the regulation of centrosome localized proteins such as NEK7 and HDAC6, however, whether the centrosome composition or structure is altered during the inflammasome activation is not known. Our data show that levels of the centrosomal scaffold protein pericentrin (PCNT) are reduced upon NLRP3 inflammasome activation via different activators in human and murine macrophages. PCNT loss occurs in the presence of membrane stabilizer punicalagin, suggesting this is not a consequence of membrane rupture. We found that PCNT loss is dependent on NLRP3 and active caspases as MCC950 and pan caspase inhibitor ZVAD prevent its degradation. Moreover, caspase-1 and GSDMD are both required for this NLRP3-mediated PCNT loss because absence of caspase-1 or GSDMD triggers an alternative regulation of PCNT via its cleavage by caspase-3 in response to nigericin stimulation. PCNT degradation occurs in response to nigericin, but also other NLRP3 activators including lysomotropic agent L-Leucyl-L-Leucine methyl ester (LLOMe) and hypotonicity but not AIM2 activation. Our work reveals that the NLRP3 inflammasome activation alters centrosome composition highlighting the need to further understand the role of this organelle during inflammatory responses.

Studies on Autophagy and Apoptosis of Fibrosarcoma HT-1080 Cells Mediated by Chalcone with Indole Moiety.

This study demonstrated the anticancer efficacy of chalcones with indole moiety (MIPP, MOMIPP) in fibrosarcoma cells for the first time. The results showed that MIPP and MOMIPP reduced the viability of HT-1080 cells in a concentration-dependent manner. MOMIPP was more active than MIPP in HT-1080 cells, showing lower IC50 values (3.67 vs. 29.90 μM). Both compounds at a concentration of 1 μM induced apoptosis in HT-1080 cells, causing death strictly related to caspase activation, as cell viability was restored when the caspase inhibitor Z-VAD was added. Reactive oxygen species production was approximately 3-fold higher than in control cells, and cotreatment with the inhibitor of mitochondrial ATPase oligomycin diminished this effect. Such effects were also reflected in mitochondrial dysfunction, including decreased membrane potential. Interestingly, the compounds that were studied caused massive vacuolization in HT-1080 cells. Immunocytochemical staining and TEM analysis showed that HT-1080 cells exhibited increased expression of the LC3-II protein and the presence of autophagosomes with a double membrane, respectively. Both compounds induced apoptosis, highlighting a promising link between autophagy and apoptosis. This connection could be a new target for therapeutic strategies to overcome chemoresistance, which is a significant cause of treatment failure and tumour recurrence in fibrosarcoma following traditional chemotherapy.

The type III secretion system effector EspO of enterohaemorrhagic Escherichia coli inhibits apoptosis through an interaction with HAX-1.

Many enteric pathogens employ a type III secretion system (T3SS) to translocate effector proteins directly into the host cell cytoplasm, where they subvert signalling pathways of the intestinal epithelium. Here, we report that the anti-apoptotic regulator HS1-associated protein X1 (HAX-1) is an interaction partner of the T3SS effectors EspO of enterohaemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium, OspE of Shigella flexneri and Osp1STYM of Salmonella enterica serovar Typhimurium. EspO, OspE and Osp1STYM have previously been reported to interact with the focal adhesions protein integrin linked kinase (ILK). We found that EspO localizes both to the focal adhesions (ILK localisation) and mitochondria (HAX-1 localisation), and that increased expression of HAX-1 leads to enhanced mitochondrial localisation of EspO. Ectopic expression of EspO, OspE and Osp1STYM protects cells from apoptosis induced by staurosporine and tunicamycin. Depleting cells of HAX-1 indicates that the anti-apoptotic activity of EspO is HAX-1 dependent. Both HAX-1 and ILK were further confirmed as EspO1-interacting proteins during infection using T3SS-delivered EspO1. Using cell detachment as a proxy for cell death we confirmed that T3SS-delivered EspO1 could inhibit cell death induced during EPEC infection, to a similar extent as the anti-apoptotic effector NleH, or treatment with the pan caspase inhibitor z-VAD. In contrast, in cells lacking HAX-1, EspO1 was no longer able to protect against cell detachment, while NleH1 and z-VAD maintained their protective activity. Therefore, during both infection and ectopic expression EspO protects cells from cell death by interacting with HAX-1. These results suggest that despite the differences between EHEC, C. rodentium, Shigella and S. typhimurium infections, hijacking HAX-1 anti-apoptotic signalling is a common strategy to maintain the viability of infected cells.

Combined Effect of Caspase-Dependent and Caspase-Independent Apoptosis in the Anticancer Activity of Gold Complexes with Phosphine and Benzimidazole Derivatives.

Since the potential anticancer activity of auranofin was discovered, gold compounds have attracted interest with a view to developing anticancer agents that follow cytotoxic mechanisms other than cisplatin. Two benzimidazole gold(I) derivatives containing triphenylphosphine (Au(pben)(PPh3)) (1) or triethylphosphine (Au(pben)(PEt3)) (2) were prepared and characterized by standard techniques. X-ray crystal structures for 1 and 2 were solved. The cytotoxicity of 1 and 2 was tested in human neuroblastoma SH-SY5Y cells. Cells were incubated with compounds for 24 h with concentrations ranging from 10 µM to 1 nM, and the half-maximal inhibitory concentration (IC50) was determined. 1 and 2 showed an IC50 of 2.7 and 1.6 µM, respectively. In order to better understand the type of cell death induced by compounds, neuroblastoma cells were stained with Annexin-FITC and propidium iodide. The fluorescence analysis revealed that compounds were inducing apoptosis; however, pre-treatment with the caspase inhibitor Z-VAD did not reduce cell death. Analysis of compound effects on caspase-3 activity and reactive oxygen species (ROS) production in SH-SY5Y cells revealed an antiproliferative ability mediated through oxidative stress and both caspase-dependent and caspase-independent mechanisms.

Striatin translocates to the cytosol of apoptotic cells and is proteolytically cleaved in a caspase 3-dependent manner

Striatin (STRN) is a multivalent protein holding great therapeutic potentials in view of its interaction with dynamic partners implicated in apoptosis. Although striatin-3 and striatin-4, that share high structural similarities with STRN, have been linked to apoptosis, the dynamics of STRN in apoptotic cells remain unclear. Herein, we report that the amount of STRN (110 kDa) is reduced in apoptotic cells, in response to various chemotherapeutic agents, thereby yielding a major polypeptide fragment at ~65 kDa, and three minor products at lower molecular weights. While STRN siRNA reduced the 65 kDa derivative fragment, the overexpression of a Myc-tagged STRN precipitated a novel fragment that was detected slightly higher than 65 kDa (due to the Myc-DDK tag on the cleaved fragment), confirming the cleavage of STRN during apoptosis. Interestingly, STRN cleavage was abrogated by the general caspase inhibitor Z-VAD.fmk. Cell fractionation revealed that the STRN pool, mainly distributed in the non-cytosolic fragment of naïve cells, translocates to the cytosol where it is proteolytically cleaved during apoptosis. Interestingly, the ectopic expression of caspase 3 in MCF-7 cells (deprived of caspase 3) induced STRN cleavage under apoptotic conditions. Inhibition of caspase 3 (Ac-DEVD-CHO) conferred a dose-dependent protection against the proteolytic cleavage of STRN. Collectively, our data provide cogent proofs that STRN translocates to the cytosol where it undergoes proteolytic cleavage in a caspase 3-dependent manner during apoptosis. Thus, this study projects the cleavage of STRN as a novel marker for apoptosis to serve pharmacological strategies targeting this particular form of cell death.

Novel nickel(II), palladium(II), and platinum(II) complexes having a pyrrolyl-iminophosphine (PNN) pincer: Synthesis, crystal structures, and cytotoxic activity

A pyrrolyl-iminophosphine (PNNH) which would act as a potential terdentate ligand has been prepared by Schiff base reaction. Complexes [M(PNN)X] (M = Ni; X = Cl (1), Pd; X = Cl (2), Br (3), I (4), M = Pt; X = Cl (5)) were prepared. The title complexes were characterized by various spectroscopic (IR, 1H, 13C, and 31P NMR) and elemental analyses. The molecular structures of 1, 2, and 5 have been established by single-crystal X-ray crystallography, demonstrating a distorted square planar geometry comprising two 5-membered metallacyclic rings. Complexes 1 and 2 were found to crystallize in the orthorhombic while complex 5 crystallizes in the monoclinic. Cytotoxicities of the complexes along with PNNH were evaluated against A549 (lung), SK-OV-3 (ovarian), SM-MEL-2 (skin), and HCT15 (colon) human cancer cell lines by sulforhodamine B assay. Notably, the palladium(II) complex (2) shows the highest activity. Apoptosis activity along with the caspase inhibitor Z-VAD (Z-Val-Ala-Asp-fluoromethyl ketone) assay of 2 and 5 against A549 and HCT15 cancer cell lines were investigated to learn a mechanistic pathway for the observed cytotoxicity, practically eliminating an apoptotic cell-death route. Complexes 2 and 5 were studied to DNA cleavage assay and molecular docking simulation. The DNA (pcDNA3.0) cleavage experiment evaluates complex 5 interacting with DNA, more effectively, in comparison to complex 2. Molecular docking simulation of 2 and 5 toward DNA and GRP78 (glucose-regulated protein 78) was performed to predict binding sites of ligand-receptors and a plausible mechanistic aspect of metallodrug-action.

Crucial role of the terminal complement complex in chondrocyte death and hypertrophy after cartilage trauma

Innate immune response and particularly terminal complement complex (TCC) deposition are thought to be involved in the pathogenesis of posttraumatic osteoarthritis. However, the possible role of TCC in regulated cell death as well as chondrocyte hypertrophy and senescence has not been unraveled so far and was first addressed using an ex vivo human cartilage trauma-model. Cartilage explants were subjected to blunt impact (0.59J) and exposed to human serum (HS) and cartilage homogenate (HG) with or without different potential therapeutics: RIPK1-inhibitor Necrostatin-1 (Nec), caspase-inhibitor zVAD, antioxidant N-acetyl cysteine (NAC) and TCC-inhibitors aurintricarboxylic acid (ATA) and clusterin (CLU). Cell death and hypertrophy/senescence-associated markers were evaluated on mRNA and protein level. Addition of HS resulted in significantly enhanced TCC deposition on chondrocytes and decrease of cell viability after trauma. This effect was potentiated by HG and was associated with expression of RIPK3, MLKL and CASP8. Cytotoxicity of HS could be prevented by heat-inactivation or specific inhibitors, whereby combination of Nec and zVAD as well as ATA exhibited highest cell protection. Moreover, HS+HG exposition enhanced the gene expression of CXCL1, IL-8, RUNX2 and VEGFA as well as secretion of IL-6 after cartilage trauma. Our findings imply crucial involvement of the complement system and primarily TCC in regulated cell death and phenotypic changes of chondrocytes after cartilage trauma. Inhibition of TCC formation or downstream signaling largely modified serum-induced pathophysiologic effects and might therefore represent a therapeutic target to maintain the survival and chondrogenic character of cartilage cells.

Curcumin Enhances the Sensitivity of Bortezomib By Inhibiting NF-Κb in ABC Diffuse Large B-Cell Lymphoma Cells

Background Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-hodgkin lymphoma with great heterogeneity in clinical behavior and response to treatment. According to gene expression profile, DLBCL can be divided into at least two subtypes: germinal center B cell type (GCB) and activated B cell type (ABC). ABC-type DLBCL is commonly activated by the NF-κB pathway, but the proteasome inhibitor bortezomib does not significantly improve the prognosis of ABC-DLBCL. Curcumin inhibit the proliferation of various tumor cells by inhibiting the activation of NF-κB. Our purpose was to evaluate whether curcumin could enhance the ensitivity of Bortezomib in ABC-DLBCL. Methods MTT assay was used to evaluate the proliferation of 2 ABC-DLBCL cell lines by treated with curcumin and bortezomib. Apoptosis were detected by FCM after staining with Annexin V/SYTOX Green.Western Blot was used to evaluated the expression of PARP, NF-κB, I κBα/p-IκBα and caspase-3 in ABC-DLBCL cells treated with curcumin and bortezomib. Results Both curcumin and bortezomib could inhibit the proliferation and induce apoptosis in ABC-DLBCL cell lines. Curcumin decreased the expression of p-IκBα, NF-κB/p65 and increased the expression of Cleaved PARP in ABC-DLBCL cell lines. Caspase Inhibitor Z-VAD could reverse the curcumin induced proliferation inhibition and apoptosis by decreasing the cleaved PARP expression. The combination of curcumin and bortezomib could further enhance the proliferation inhibition and apoptosis in ABC-DLBCL cell lines by inhibiting NF-κB. Conclusions Curcumin induced proliferation inhibition and apoptosis by inhibiting NF-κB in ABC-DLBCL. It may sensitize ABC-DLBCL cell lines to the cytotoxic effects of bortezomib by inhibiting NF-κB. Disclosures No relevant conflicts of interest to declare.

Evidence of necroptosis in osteoarthritic disease: investigation of blunt mechanical impact as possible trigger in regulated necrosis

Joint injuries are highly associated with cell death and development of posttraumatic osteoarthritis (PTOA). The present study focused on necroptosis as a possible modality of chondrocyte death after cartilage trauma and its relevance in OA disease in general. For this purpose, apoptosis- and necroptosis-associated markers were determined in highly degenerated (ICRS ≥ 3) as well as macroscopically intact cartilage tissue (ICRS ≤ 1) by means of real-time PCR and immunohistochemistry (IHC). Moreover, influence of blunt trauma and/or stimulation with cycloheximide (CHX), TNF-a, and caspase-inhibitor zVAD were investigated in cartilage explants (ICRS ≤ 1). Further characterization of necroptosis was performed in isolated chondrocytes. We found that gene expression levels of RIPK3 (4.2-fold, P < 0.0001) and MLKL (2.7-fold, P < 0.0001) were elevated in highly degenerated cartilage tissue, which was confirmed by IHC staining. After ex vivo trauma and/or CHX/TNF stimulation, addition of zVAD further enhanced expression of necroptosis-related markers as well as release of PGE2 and nitric oxide, which was in line with increased cell death and subsequent release of intracellular HMGB1 and dsDNA in CHX/TNF stimulated chondrocytes. However, trauma and/or chemically induced cell death and subsequent release of pro-inflammatory mediators could be largely attenuated by RIPK1-inhibitor necrostatin 1 or antioxidant N-acetylcysteine. Overall, the study provided clear evidence of necroptotic cell death in OA disease. Moreover, a possible link between cartilage injury and necroptotic processes was found, depending on oxidative stress and cytokine release. These results contribute to further understanding of cell death in PTOA and development of novel therapeutic approaches.

Decrease in fat de novo synthesis and chemokine ligand expression in non-alcoholic fatty liver disease caused by inhibition of mixed lineage kinase domain-like pseudokinase.

Receptor-interacting serine/threonine kinase 3 and mixed lineage kinase domain-like pseudokinase (MLKL) have gained attention as apoptosis alternate cell death signaling molecules. We aimed to evaluate the role of MLKL in non-alcoholic fatty liver disease (NAFLD). Hepatic tissue MLKL expression was compared between NAFLD patients and healthy controls. High-fat diet was fed to wild-type and MLKL-knockout (KO) mice for 12weeks. Brown adipose fat tissue was measured by [18 F]-fluorodeoxyglucose positron emission tomography. Energy expenditure was measured by indirect calorimetry. Anti-MLKL effects were also evaluated in in vitro setting using U937 and HepG2 cells. Hepatic tissue MLKL expression increased in NAFLD patients compared with healthy controls. MLKL expression increased according to the degree of steatosis, ballooning, and inflammation. High-fat diet-fed MLKL-KO mice displayed decreased alanine aminotransferase, triglycerides, liver weight, NAFLD activity score (6.3 vs 3.5, P<0.001), steatosis score (3.0 vs 1.8, P<0.001), inflammation, and ballooning degeneration compared with wild-type mice. SREBP1c, fatty acid synthase, and SCD-1 expressions decreased in MLKL-KO mice. Adipose tissue F4/80-positive crown-like structures were also reduced in MLKL-KO mice. HepG2 cells treated with necrosulfonamide (an MLKL inhibitor) showed reduced Nile red staining and reduced SREBP1c and SCD-1 expressions. Stimulation of necroptosis using lipopolysaccharide+caspase inhibitor (zVAD) increased CXCL1/2 expressions in U937 monocyte cells. Lipopolysaccharide+zVAD-induced increased expressions of CXCL1/2 were reduced with necrosulfonamide treatment. Mixed lineage kinase domain-like pseudokinase inhibition has protective effects in non-alcoholic steatohepatitis by decreasing hepatic de novo fat synthesis and chemokine (C-X-C motif) ligand expressions.

Assessing the safety of complementary medicines in lactation

A pyrrolyl-iminophosphine (PNNH) which would act as a potential terdentate ligand has been prepared by Schiff base reaction. Complexes [M(PNN)X] (M = Ni; X = Cl (1), Pd; X = Cl (2), Br (3), I (4), M = Pt; X = Cl (5)) were prepared. The title complexes were characterized by various spectroscopic (IR, 1H, 13C, and 31P NMR) and elemental analyses. The molecular structures of 1, 2, and 5 have been established by single-crystal X-ray crystallography, demonstrating a distorted square planar geometry comprising two 5-membered metallacyclic rings. Complexes 1 and 2 were found to crystallize in the orthorhombic while complex 5 crystallizes in the monoclinic. Cytotoxicities of the complexes along with PNNH were evaluated against A549 (lung), SK-OV-3 (ovarian), SM-MEL-2 (skin), and HCT15 (colon) human cancer cell lines by sulforhodamine B assay. Notably, the palladium(II) complex (2) shows the highest activity. Apoptosis activity along with the caspase inhibitor Z-VAD (Z-Val-Ala-Asp-fluoromethyl ketone) assay of 2 and 5 against A549 and HCT15 cancer cell lines were investigated to learn a mechanistic pathway for the observed cytotoxicity, practically eliminating an apoptotic cell-death route. Complexes 2 and 5 were studied to DNA cleavage assay and molecular docking simulation. The DNA (pcDNA3.0) cleavage experiment evaluates complex 5 interacting with DNA, more effectively, in comparison to complex 2. Molecular docking simulation of 2 and 5 toward DNA and GRP78 (glucose-regulated protein 78) was performed to predict binding sites of ligand-receptors and a plausible mechanistic aspect of metallodrug-action.

Morniga-G, a T/Tn-Specific Lectin, Induces Leukemic Cell Death via Caspase and DR5 Receptor-Dependent Pathways.

Morniga-G, the Gal-specific black mulberry (Morus nigra) lectin, displays high affinity for T (CD176) and Tn (CD175) antigens, frequently expressed at the cancer cell surface. The effects of Morniga-G were investigated on a Tn-positive leukemic Jurkat cell line. The lectin, used in a concentration range between 5–20 μg/mL, induced cell death in leukemic Jurkat cells. Microscopic and cytofluorometric analyses indicated that Jurkat cell death was essentially apoptotic, associated with an increase in the ceramide content and a depolarization of the mitochondrial transmembrane potential. This lectin-mediated cell death was inhibited by the pan caspase-inhibitor zVAD. In addition, cleavage of caspases 8, 9, and 3 was observed in Morniga-G-treated Jurkat cells whereas Jurkat cell lines that are deficient in caspase 8–10, caspase 9, or FADD, survived to the lectin-mediated toxicity. Furthermore, in the presence of TRAIL- or DR5-blocking mononoclonal antibodies, Jurkat cells became resistant to Morniga-G, suggesting that the lectin triggers cell death via the TRAIL/DR5 pathway. In silico computer simulations suggest that Morniga-G might facilitate both the DR5 dimerization and the building of TRAIL/DR5 complexes. Finally, upon treatment of Jurkat cells with benzyl-GalNAc, an O-glycosylation inhibitor, a decrease in Tn antigen expression associating with a reduced Morniga-G toxicity, was observed. Taken together, these results suggest that Morniga-G induces the cell death of Tn-positive leukemic cells via concomitant O-glycosylation-, caspase-, and TRAIL/DR5-dependent pathways.

Scutellaria Radix Promotes Apoptosis in Non-Small Cell Lung Cancer Cells via Induction of AMPK-Dependent Autophagy.

Scutellaria Radix (SR) is an herb traditionally used in Asian countries to treat inflammatory diseases. Recent studies report that SR exhibits anticancer activities in various types of tumors. In this study, we investigated the apoptotic and autophagic effect of SR in non-small cell lung cancer (NSCLC), the leading cause of cancer-associated death. Treatment of SR in two NSCLC cell lines, H358 and H2087 cells resulted in suppressed cell viability. Western blot assays showed increased expressions of Bcl-2-associated X protein (Bax), cleaved-caspase 3 and cleaved-Poly ADP ribose polymerase (PARP), key factors of apoptosis. Co-treatment of SR with a caspase inhibitor Z-VAD led to nullification of the antiproliferative effect, suggesting the role of apoptosis in the action mechanism of SR. Further experiments revealed autophagy was involved in the effect of SR. SR-treated NSCLC cells expressed increased ratio of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II/LC3-I. When chloroquine was co-treated with SR, this ratio was further increased, indicating SR treatment induced autophagy in NSCLC cells. Interestingly, loss of autophagy by 3-Methyladenine (3-MA) co-treatment suppressed SR-induced apoptosis. We then evaluated the relevance of AMP-activated protein kinase (AMPK) in the autophagic/apoptotic process in NSCLC by SR treatment. Immunoblot assays showed increased phosphorylation of AMPK and P70-S6 kinase in SR-treated H358 and H2087 cells. Under AMPK-inhibited conditions by compound C, SR treatment failed to induce both autophagy and apoptosis. Taken together, this study identifies the positive effect of SR in H358 and H2087 cells by inducing apoptosis via AMPK-dependent autophagy. Thus, our results suggest the potential use of SR as a novel therapeutic strategy for NSCLC patients.

Antitumor effects of β-elemene via targeting the phosphorylation of insulin receptor.

Ewing sarcoma family tumors (ESFTs) are a group of aggressive and highly metastatic tumors lacking efficient therapies. Insulin-like growth factor 1 receptor (IGF1R) blockade is one of the most efficient targeting therapy for ESFTs. However, the appliance is obstructed by drug resistance and disease recurrence due to the activation of insulin receptor (IR) signaling induced by IGF1R blockade. Herein β-elemene, a compound derived from natural plants, exhibited a remarkable proliferation repression on ESFT cells, which was weakened by a caspase inhibitor Z-VAD. β-elemene in combination with IGF1R inhibitors enhanced markedly the repression on cellular proliferation and mTOR activation by IGF1R inhibitors and suppressed the PI3K phosphorylation induced by IGF1R inhibitors. To investigate the mechanisms, we focused on the effects of β-elemene on IR signaling pathway. β-elemene significantly suppressed the insulin-driven cell growth and the activation of mTOR and PI3K in tumor cells, while the toxicity to normal hepatocytes was much lower. Further, the phosphorylation of IR was found to be suppressed notably by β-elemene specifically in tumor cells other than normal hepatocytes. In addition, β-elemene inhibited the growth of ESFT xenografts in vivo, and the phosphorylation of IR and S6 ribosomal protein was significantly repressed in the β-elemene-treated xenografts. These data suggest that β-elemene targets IR phosphorylation to inhibit the proliferation of tumor cells specifically and enhance the effects of IGF1R inhibitors. Thus, this study provides evidence for novel approaches by β-elemene alone or in combination with IGF1R blockades in ESFTs and IR signaling hyperactivated tumors.

Abstract 2428: Redox regulation of β-catenin in colorectal cancer

Abstract Colorectal cancer (CRC) is the second most newly diagnosed cancer in the United States. β-catenin is the downstream effector of the Wnt/β-catenin signaling pathway. Loss of function (LOF) mutations on adenomatous polyposis coli (APC), a critical member of the β-catenin degradation complex leads to constitutively elevated β-catenin as an early oncogenic event in 85% of CRCs and is associated with decreased CRC patient survival. Increased level of β-catenin augments the nuclear accumulation of β-catenin, where it acts a co-activator of a number of transcription factors leading to the expression of downstream target genes that promote cancer cell proliferation, survival, and migration. There is no effective therapy for β-catenin as a cause of cancer. In addition to CRC, elevated levels of Wnt signaling have been reported in leukemia, melanoma, and breast cancers highlighting the need for novel therapies that attenuate aberrant β-catenin mediated transcription. Here we report on the redox dependency of the Wnt/β-catenin signaling pathway, identified through a forward genetic screen in Drosophila, and identify thioredoxin reductase-1 (TXNR1) as a potential target for the inhibition of aberrant β-catenin transcriptional activity through in vivo studies with the APCmin/+ mouse model. Drosophila harboring a mutation in their thioredoxin reductase (trxr-1) gene and a partial lethality phenotype were used in an enhancer/suppressor forward genetic screen. Increased lethality was observed when the trxr-1 mutation (trxr-1481) was combined with heterozygous mutations and deletions in wnt2, while wnt2 deletion and mutations alone had no effect on lethality. To test the translational value of these findings, APCmin/+ mice were treated with PX-12, a human TXNR1 inhibitor, daily by gavage from day 40 to time of death. We observed a dose dependent reduction in the number and size of intestinal polyps by PX-12 treatment, and an increased survival in treated mice. Furthermore, mechanistic studies utilizing human CRC cell lines harboring LOF mutations to the β-catenin degradation complex revealed β-Catenin mediated transcription, as measured by TOP Flash luciferase assay, was diminished when TXNR1 was silenced by siRNA. Additionally, we observed decreased β-catenin protein level in CRC cell lines treated with PX-12 or siRNA targeting TXNRD1 and its functional partner thioredoxin (TXN). The addition of the pan caspase inhibitor ZVAD but not the proteasome inhibitor MG132 was able to stabilize β-catenin protein upon treatment with PX-12 suggesting β-catenin destabilization is occurring via a caspase dependent mechanism. These results suggest a conserved mechanism for the redox regulation of β-catenin involving TXNR1; furthermore, inhibition of TXNR1 activity destabilizes β-catenin protein and decreases β-catenin mediated transcription in human CRC cell lines and inhibits intestinal neoplasia development in an experimental mouse model for CRC. Citation Format: Alejandro D. Campos, Brian James, Petrus De Jong, Robert Lemos, Nikolas Marino, Garth Powis. Redox regulation of β-catenin in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2428.

Abstract 615: Absence of RIP3 Reduces Atherosclerotic Progression in Type 1 Diabetes

Introduction: Patients suffering from Type 1 diabetes (T1D) have increased risk of developing atherosclerosis. T1D is a chronic autoimmune metabolic disease characterized by a deficiency in insulin secretion and hyperglycemia, promoting oxidative stress and low-grade inflammation in part via advanced glycation end products (AGE). Recently, we and others showed that necroptosis, a newly defined pro-inflammatory programmed cell death, drives advanced atherosclerosis. Given that diabetes accelerates inflammation and atherosclerosis, we hypothesized that hyperglycemia may exacerbate macrophage necroptosis to drive atherosclerotic lesion formation. Methods/Results: Male Ldlr-/- mice were lethally irradiated, transplanted with bone-marrow from WT or Rip3 -/- mice and then administered streptozotocin (STZ) or vehicle daily for 5 days to induce T1D, prior to being fed a western diet for 6 wks. As expected, STZ-treated WT BMT Ldlr -/- mice had a marked increased in atherosclerotic aortic lesion area compared to non-diabetic mice (1.08%±0.30 vs 2.13%±0.45). which was blunted in Rip3 -/- BMT Ldr -/- mice (arch: 49% reduction, sinus: 54% reduction, p&lt;0.05). Bone marrow derived macrophages (BMDMs) cultured in high glucose (HG) increased cell death in response to necroptotic stimuli (oxidized LDL ± caspase inhibitor zVAD) relative to those cultured in low glucose (LG). Further, the addition of recombinant s100A8/9, a soluble AGE, markedly amplified oxLDL-induced death in HG cultured BMDMs with minimal effects on LG cultured BMDMs, suggesting that pro-inflammatory AGEs released in diabetes can augment macrophage cell death. Further, the increased cell death induced by oxLDL+S100A8/9 was inhibited by a necroptotic inhibitor, Nec-1, or in Rip3 -/- BMDMs cultured in HG. Mechanistically, treatment of BMDMs cultured in HG with oxLDL+s100A8/9 increased the expression of Rip3 and Mlkl (~5-fold and ~2-fold respectively), which may drive necroptotic cell death during hyperglycemia. Conclusion: Deletion of Rip3 markedly halts atherosclerotic progression in a STZ-induced T1D mouse model by inhibiting macrophage necroptosis induced by both atherogenic ligands (oxLDL) and soluble AGE products (s100A8/9).

The release of microparticles and mitochondria from RAW 264.7 murine macrophage cells undergoing necroptotic cell death in vitro

Microparticles (MPs) are small membrane-bound vesicles released from activated or dying cells. As shown previously, LPS stimulation of the RAW 264.7 macrophage cell line can induce MP release, with the caspase inhibitor Z-VAD increasing the extent of this process. Since combined treatment of cells with LPS and Z-VAD can induce necroptosis, we explored particle release during this form of cell death using flow cytometry to assess particle size, binding of annexin V and staining for DNA with propidium iodide (PI) and SYTO 13. The role of necroptosis was assessed by determining the effects of necrostatin, an inhibitor of RIP1, a kinase regulating this form of cell death. These studies demonstrated that, during necroptosis, RAW 264.7 cells release MPs that resemble those released from cells treated with staurosporine to induce apoptosis. The particles contained DNA as determined by binding of PI and SYTO 13, with PCR analysis demonstrating both chromosomal and mitochondrial DNA. The presence of mitochondria in the MP preparations was demonstrated by staining with MitoTracker Green. Flow cytometry indicated that purified mitochondria have properties of MPs. Together, these studies indicate that cells undergoing necroptosis can release MPs and that mitochondria can be components of MP preparations.

Use of Lentiviral Particles As a Cell Membrane-Based mFasL Delivery System for In Vivo Treatment of Inflammatory Arthritis.

During budding, lentiviral particles (LVP) incorporate cell membrane proteins in the viral envelope. We explored the possibility of harnessing this process to generate LVP-expressing membrane proteins of therapeutic interest and studied the potential of these tools to treat different pathologies. Fas-mediated apoptosis is central to the maintenance of T cell homeostasis and prevention of autoimmune processes. We prepared LVP that express murine FasL on their surface. Our data indicate that mFasL-bearing LVP induce caspase 3 and 9 processing, cytochrome C release, and significantly more cell death than control LVP in vitro. This cytotoxicity is blocked by the caspase inhibitor Z-VAD. Analysis of the application of these reagents for the treatment of inflammatory arthritis in vivo suggests that FasL-expressing LVP could be useful for therapy in autoimmune diseases such as rheumatoid arthritis, where there is an excess of Fas-expressing activated T cells in the joint. LVP could be a vehicle not only for mFasL but also for other membrane-bound proteins that maintain their native conformation and might mediate biological activities.

RETRACTED ARTICLE: RIPK3 interactions with MLKL and CaMKII mediate oligodendrocytes death in the developing brain

Oligodendrocyte progenitor cells (OPCs) death is a key contributor to cerebral white matter injury (WMI) in the developing brain. A previous study by our group indicated that receptor-interacting proteins (RIPs) are crucial in mediating necroptosis in developing neurons. However, whether this mechanism is involved in OPCs death is unclear. We aimed to explore the mechanisms of RIP-mediated oligodendrocytes (OLs) death in the developing brain. Oligodendrocytes necroptosis was induced by oxygen-glucose deprivation plus caspase inhibitor zVAD treatment (OGD/zVAD) in vitro. Western blotting and immunofluorescence were used to detect RIPK1, RIPK3, mixed lineage kinase domain-like protein (MLKL), and Ca2+ and calmodulin-dependent protein kinase IIδ (CaMKIIδ). Immunoprecipitation was used to assess the interactions between RIPK3 and RIPK1, MLKL, and CaMKIIδ. Necrostatin-1 was used to disturb the RIPK3–RIPK1 interaction, and siRNA was used to inhibit RIPK3 or MLKL expression. Oligodendrocytes death was examined using PI staining, EM, and cell membrane leakage assays. In vivo, brain damage in neonatal rats was induced by hypoxia–ischemia (HI). This was followed by an examination of myelin development. We found that OGD/zVAD treatment upregulates the expression of RIPK3 and the interaction of RIPK3 with RIPK1, MLKL, and CaMKIIδ. Inhibition of the RIPK3-MLKL or RIPK3-CaMKIIδ interaction attenuates OLs death induced by OGD/zVAD. These protective mechanisms involve the translocation of MLKL to the OLs membrane, and the phosphorylation of CaMKIIδ. However, inhibition of the RIPK3–RIPK1 interaction did not protect OLs death induced by OGD/zVAD. In vivo studies indicated that the disrupted development of myelin was attenuated after the inhibition of RIPK3-MLKL or RIPK3-CaMKIIδ interaction. Taken together, our data indicate that RIPK3 is a key factor in protection against OLs death and abnormal myelin development via its interaction with MLKL and CaMKIIδ after HI. This suggests that RIPK3 may be a potential target for the treatment of WMI in neonates.

Camalexin-Induced Cell Membrane Scrambling and Cell Shrinkage in Human Erythrocytes

Background/Aims: The thaliana phytoalexin Camalexin has been proposed for the treatment of malignancy. Camalexin counteracts tumor growth in part by stimulation of suicidal death or apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms contributing to the complex machinery executing eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), oxidative stress, ceramide, protein kinase C and caspases. The present study explored, whether Camalexin induces eryptosis and, if so, to shed light on mechanisms involved. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo-3 fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to Camalexin significantly increased the percentage of annexin-V-binding cells (≥ 10 µg/ml), significantly decreased forward scatter (≥ 5 µg/ml) and significantly increased Fluo-3-fluorescence (≥ 10 µg/ml), but did not significantly modify DCFDA fluorescence or ceramide abundance. The effect of Camalexin on annexin-V-binding was significantly blunted by removal of extracellular Ca²⁺, by kinase inhibitors staurosporine (1 µM) and chelerythrine (10 µM), as well as by caspase inhibitors zVAD (10 µM) and zIETD-fmk (50 µM). Conclusions: Camalexin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part depending on Ca²⁺ entry, as well as staurosporine and chelerythrine sensitive kinase(s) as well as zVAD and zIETD-fmk sensitive caspase(s).