Presence Of Caspase Inhibitor Research Articles

Effect of miltirone on proliferation and apoptosis of leukemia THP-1 cells

To investigate the toxicity and related mechanism of miltirone to human acute myeloid leukemia THP-1 cells. To be specific, the active components and targets of miltirone were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), and the target proteins were converted into standard gene names with UniProt. Acute leukemia-rela-ted target genes were screened from GeneCards and DisGeNET. Venn diagram was constructed with Venny 2.1 to yield the common targets of the disease and the drug. The protein-protein interaction(PPI) network was constructed by STRING and Cytoscape 3.8.2. THP-1 cells in the logarithmic growth phase were treated with dimethyl sulfoxide(DMSO), and 2.5, 5, 10, 15, and 20 μmol·L~(-1) miltirone for 24 h, respectively. The proliferation rate of cells was analyzed by carboxyfluorescein diacetate succinimidyl ester(CFSE), apoptosis rate by flow cytometry with Annexin V-PE/7 AAD staining, and cell morphology by acridine orange staining. Real-time quantitative PCR(qPCR) was employed to detect the mRNA levels of nuclear receptor coactivator 2(NCOA2), poly(ADP-ribose) polymerase-1(PARP1), B-cell lymphoma-2(Bcl-2)-associated X protein(Bax), Bcl-2, and cysteine aspartyl protease-3(caspase-3). The effect of miltirone on apoptosis was detected in presence of caspase inhibitor Z-VAD-FMK. A total of 26 targets of miltirone, 1 046 genes related to acute leukemia, and 6 common targets of the two were screened out. Flow cytometry result showed miltirone at 10 μmol·L~(-1) can inhibit proliferation and promote apoptosis of THP-1 cells. The typical manifestations of apoptosis, such as cell shrinkage, nuclear rupture, and chromatin agglomerate were displayed by acridine orange staining. The decreased mRNA levels of NCOA2 and PARP1 and increased Bax/Bcl-2 ratio and the activity of pro-apoptotic protein caspase-3 were observed. Z-VAD-FMK can attenuate the apoptosis-inducing effect of miltirone. This study indicates that miltirone can inhibit the proliferation and promote the apoptosis of THP-1 cells, by down-regulating NCOA2 and PARP1, raising Bax/Bcl-2 ratio, and activating caspase-3.

Acceleration of slow autophagy flux induced by arabinofuranosyl cytidine improves its antileukemic effectiveness in M-NFS-60 cells.

Arabinofuranosyl cytidine (AraC) is an analog of deoxycytidine used as an anticancer drug for leukemic patients. The effective dose always produces severe complications. The present study investigated the modulation of autophagy and its impact on the cytotoxicity of AraC toward murine myelogenous leukemia cells (M-NFS-60). Autophagy was inhibited by NH4Cl or Bafilomycin A1 or enhanced by amino acid starvation, glucose starvation, mild hyperthermia (41 °C), or rapamycin (Rap). Cells were treated with different concentrations, 0 to 2 μM, of AraC in the presence or absence of autophagy modulators. AraC-induced apoptosis is combined with autophagy, especially at lower concentrations. This autophagy is characterized by a slow flux, as indicated by levels of LC3B II and P62 proteins. Inhibition of autophagy did not alter cleaved caspase 3 levels (c-casp.3) or cell viability measured by MTT assays. Conversely, acceleration of AraC-induced autophagy by co-treatment with autophagy inducers reduced cell viability and increased c-casp.3 and c-PARP levels. Further, c-PARP levels were reduced in the presence of caspase inhibitor, Z-VAD-FMK. Enhancement of slow autophagic flux induced by low concentrations of AraC significantly increased the cytotoxicity of AraC toward M-NFS-60 cells. Such coadministration of autophagy inducers might improve the efficacy of AraC treatment and reduce effective doses.

Nuclear Opening and Histone Release Are Essential for Nuclear but Not Chromatin Condensation during Terminal Erythropoiesis

Nuclear condensation and enucleation are characteristic processes in mammalian terminal erythropoiesis. These processes are associated with the transient nuclear opening formation that mediates partial histone release to the cytoplasm. Our previous report showed that caspases are involved in the cleavage of nuclear lamina to enable histone release. However, it remains unclear whether nuclear opening formation and histone release regulate the genomic three-dimensional organization during nuclear condensation. To answer this question, we cultured E13.5 mouse fetal liver Ter119 negative erythroid progenitor cells in erythropoietin (EPO) containing medium for 48 h with or without the presence of caspase inhibitor. As expected, caspase inhibitor blocked nuclear opening formation and histone release, and significantly reduced nuclear condensation and enucleation. We next performed a Hi-C sequencing to investigate chromatin structural change during terminal differentiation and nuclear condensation. To this end, the cultured fetal liver erythroid cells with or without caspase inhibitor were harvested at 30 h right before enucleation for Hi-C sequencing. The sequencing results showed that cells at 30 h contain significantly more interactions than freshly isolated erythroid progenitors, which is consistent with chromatin condensation during terminal erythropoiesis. Further analysis showed that increased interactions mainly accumulate as inter-chromosomal interactions, suggesting inter-chromosome interaction is the dominant structural force driving erythrocyte chromatin condensation. Surprisingly, there were no significant chromatin structural changes between caspase inhibitor treated and mock-treated cells when compared at 30 h. We also performed ATAC-seq and RNA-seq with the same experiment settings, both corresponded to Hi-C sequencing and showed little difference under caspase inhibitor treatment. These results indicate that although histone release and nuclear condensation are compromised with the inhibition of caspases, chromatin stays condensed with well-organized three-dimensional structure and appropriate gene expression regulations.To further confirm this phenomenon, we generated caspase-3 and -7 double knock out (cas3cas7-/-) mice. Cas3cas7-/- mice are embryonically lethal due to defective cardiac development. The hematopoietic tissues in these mice have not been well studied. We harvested fetal liver Ter119 negative erythroid progenitor cells from E13.5 cas3cas7-/- mice and the cells from the littermate (cas7-/-, cas3+/-cas7-/-) mice were used as controls. We first cultured Ter119 negative fetal liver erythroid progenitors in EPO containing medium for 48 h. Immunofluorescence analysis showed that the nuclear opening was significantly inhibited, and the nuclear size significantly increased in the erythroid cells from cas3cas7-/- mice due to failure of histone release into cytoplasm. Flow cytometry analysis showed that enucleation was significantly impaired in cas3cas7-/- cells, but the cells could still differentiate although with lower efficiency. We further performed an in vivo assay in which E13.5 cas3cas7-/- fetal liver cells were transplanted into wild type lethally irradiated recipient mice. EPO medium cultured bone marrow lineage negative cells from these transplanted mice showed significant reduction in nuclear opening and histone release, and enlargement of nuclear size. However, these mice survived well despite anemia. These results indicate a portion of orthochromatic erythroblasts managed to enucleate even with the less condensed nuclei.Overall, our study demonstrates that nuclear opening and histone release are essential for nuclear condensation but have minimal effects on chromatin condensation or the regulation of gene expression in terminal erythropoiesis. Appropriate nuclear condensation is important for efficient enucleation. However, orthochromatic erythroblasts could still manage to enucleate although with low efficacies. DisclosuresNo relevant conflicts of interest to declare.

Biphasic Increases of Cell Surface Calreticulin Following Treatment with Mitoxantrone.

Calreticulin (CRT) and calnexin (CNX), homologous major chaperones in the endoplasmic reticulum (ER), are known to translocate to the cell surface in response to chemotherapeutic agents, such as mitoxantrone (MIT), and cellular stresses, including apoptosis. Cell surface CRT (ecto-CRT) is relevant to the phagocytic uptake of cancer cells and dying cells, and pre-apoptotic exposure of CRT has been reported to result in enhanced immunogenicity of dying tumor cells, serving as a damage-associated molecular pattern (DAMP). In this study, HT-29 cells were treated with MIT to induce ER stress, and ecto-CRT and cell surface CNX were quantified by flow cytometry in the absence or presence of caspase inhibitors, a calpain inhibitor, or a scavenger of reactive oxygen species. The biphasic (early transient and late sustained) increase of ecto-CRT on HT-29 cells was observed after treatment with MIT. We confirmed that the early increase in ecto-CRT after 4 h of MIT treatment was not related to apoptosis, whereas the increase of ecto-CRT, as well as that of cell-surface CNX, during the later stage of treatment was caspase dependent and related to apoptosis. In addition, our results suggested that the early peak of ecto-CRT was mediated by activation of caspase 8 by ER stress. Thus, the physiological significance of the late increases in cell-surface CRT and/or CNX might be considered an "eat-me signal" for the removal of dead cells by phagocytosis, while the early increase in ecto-CRT caused by ER stress might enhance the immunogenicity of stressed tumor cells.

Apoptotic Fragmentation of Tricellulin.

Apoptotic extrusion of cells from epithelial cell layers is of central importance for epithelial homeostasis. As a prerequisite cell–cell contacts between apoptotic cells and their neighbors have to be dissociated. Tricellular tight junctions (tTJs) represent specialized structures that seal polarized epithelial cells at sites where three cells meet and are characterized by the specific expression of tricellulin and angulins. Here, we specifically addressed the fate of tricellulin in apoptotic cells. Methods: Apoptosis was induced by staurosporine or camptothecin in MDCKII and RT-112 cells. The fate of tricellulin was analyzed by Western blotting and immunofluorescence microscopy. Caspase activity was inhibited by Z-VAD-FMK or Z-DEVD-FMK. Results: Induction of apoptosis induces the degradation of tricellulin with time. Aspartate residues 487 and 441 were identified as caspase cleavage-sites in the C-terminal coiled-coil domain of human tricellulin. Fragmentation of tricellulin was inhibited in the presence of caspase inhibitors or when Asp487 or Asp441 were mutated to asparagine. Deletion of the tricellulin C-terminal amino acids prevented binding to lipolysis-stimulated lipoprotein receptor (LSR)/angulin-1 and thus should impair specific localization of tricellulin to tTJs. Conclusions: Tricellulin is a substrate of caspases and its cleavage in consequence contributes to the dissolution of tTJs during apoptosis.

Induction of apoptosis by fucoidan isolated from a traditional food, Saccharina longissima, in U937 human leukemia cells

Background: A brown seaweed, Saccharina longissima (Laminaria angustata var. longissima) has been consumed in Okinawa for 300 years. It is native to the southernmost part of Japan, in Okinawa, but grows on the Pacific coast (Kushiro and Nemuro) in Hokkaido in the northernmost part of Japan. Previously, we reported the structural characteristics from S. longissima. The fucoidan consisted of L-fucose, D-galactose, D-glucose, D-xylose, D-glucuronic acid, and sulfuric acid. This fucoidan activated the RAW 264.7 murine macrophage cell line. The aim of this study was to investigate the effects of a fucoidan isolated from Saccharina longissima on cell anti-proliferation and apoptosis in the U937 human leukemia monocyte lymphoma cell line. Methods: U937 cells were incubated with four fucoidan concentrations. The degree of apoptosis was determined using the APOPercentage APOPtosis assay, caspaer-3 activity assay and Western blotting analysis assay. Results: The treatment of cell with a fucoidan highly substituted with sulfate, in addition to L-fucose residues, inhibited cell growth. Apoptosis increased in U937 cells in a dose-dependent manner following the addition of fucoidan. However, the fucoidan did not induce apoptosis in the presence of caspase inhibitor (z-VAD-FMK).Conclusions: The fucoidan from Saccharina longissima induced apoptosis in U937 cells. Accordingly, the kelp containing this fucoidan may contribute to the observed longevity of the Okinawan population.Keywords: fucoidan, Saccharina longissimi, brown seaweed, traditional food, human leukemia cells, apoptosis

Histone H4 is cleaved by granzyme A during staurosporine-induced cell death in B-lymphoid Raji cells.

Granzyme A (GzmA) was first identified as a cytotoxic T lymphocyte protease protein with limited tissue expression. A number of cellular proteins are known to be cleaved by GzmA, and its function is to induce apoptosis. Histones H1, H2B, and H3 were identified as GzmA substrates during apoptotic cell death. Here, we demonstrated that histone H4 was cleaved by GzmA during staurosporine-induced cell death; however, in the presence of caspase inhibitors, staurosporine-treated Raji cells underwent necroptosis instead of apoptosis. Furthermore, histone H4 cleavage was blocked by the GzmA inhibitor nafamostat mesylate and by GzmA knockdown using siRNA. These results suggest that histone H4 is a novel substrate for GzmA in staurosporine-induced cells. [BMB Reports 2016; 49(10): 560-565]

FOXO1 Activation Is an Effector of SYK and AKT Inhibition in Tonic BCR Signal-Dependent Diffuse Large B-Cell Lymphomas

Introduction: In normal B lymphocytes, B-cell receptor (BCR)-induced activation of PI3K-AKT kinases and subsequent inactivation of FOXO1 is a critical pro-survival component of tonic BCR signaling. In murine models, conditional deletion of FOXO1 protected quiescent peripheral B cells from apoptosis mediated by inducible loss of the BCR, demonstrating that PI3K-AKT-FOXO1 axis plays a central role in B-cell homeostasis. Disruption of the BCR signaling by SYK inhibitor leads also to the apoptosis of BCR-dependent DLBCLs, at least in part via a mechanism involving decreased activity of PI3K/AKT axis. Herein, we investigated the role of FOXO1 in toxicity of BCR pathway/SYK inhibition in human BCR-dependent lymphomas.Methods: BCR-dependent DLBCL cell lines (DHL4, DHL6, Ly1, Ly7) were incubated with SYK inhibitor R406 (4 µM) and AKT phosphorylation, FOXO1 activation and transcriptional activity were assessed by phospho-specific flow cytometry, Western Blot and qPCR. Toxicity of active FOXO1 was assessed by transducing DLBCL cells with a constitutively nuclear FOXO1 mutant. FOXO1 knock-down in DLBCL cell was achieved with shRNA. DREAM cleavage and HRK expression were assessed by Western Blot and qPCR, respectively, in the absence or presence of caspase inhibitors. DLBCL cell viability and apoptosis after incubation with SYK and/or AKT inhibitors R406 and MK2206, were assessed by MTS assay and Annexin V/PI staining. Drug interactions were quantitated by CompuSyn software. The expression of p-SYK and FOXO1 in DLBCL samples was assessed by immunohistochemistry (IHC) in 60 DLBCL patients.Results: Since FOXO1 is a major effector of tonic BCR signaling, we assessed the activity of FOXO1 in DLBCL cells after SYK inhibition. In all tested cell lines, AKT and FOXO1 phosphorylations decreased after incubation with SYK inhibitor. Diminished FOXO1 phosphorylation resulted in its nuclear relocalization and induction of FOXO1-dependent expression of p27, BIM, TRAIL and GADD45A. To assess whether the increased activity of FOXO1 is sufficient to induce apoptosis of DLBCL cells, we transduced DHL4 cells with a constitutively nuclear and transcriptionally active FOXO1-3A mutant. The mutant induced G1/S cell cycle arrest and triggered apoptosis, whereas wild-type FOXO1 did not change proliferation or cellular viability, demonstrating that FOXO1 activation is sufficient to induce apoptosis of DLBCL cells. Next, we assessed the toxicity of SYK inhibitor in cells lacking FOXO1. Cells with depleted FOXO1 exhibited 70% lower sensitivity to SYK inhibitor than control cells (p<0.0001). Since in previous studies expression of the proapoptotic member of BCL2 family, HRK, was required for SYK-inhibitor induced cell death in DLBCL cells, we determined the role of FOXO1 activation in HRK expression. HRK expression was dramatically increased in SYK-inhibitor treated control cells, but not in FOXO1-deficient cells. Consistent with this, SYK inhibitor triggered cleavage of HRK transcriptional repressor DREAM only in control cells, but not in FOXO1-depleted cells. HRK induction was blocked by caspase inhibitors. Since AKT is major kinase regulating both FOXO1 activity and HRK induction, we assessed the combined effects of the AKT inhibitor MK-2206 with R406 and found markedly synergistic toxicity (combination index [CI] 0.5-0.8). Combination of inhibitors in FOXO1-depleted cells did not trigger cell death, highlighting the critical effector role of FOXO1. FOXO1 expression was present in 80% of primary DLBCL samples and correlated with SYK activity (p=0.009). High levels of FOXO1 protein expression were associated with longer OS (log rank, p=0.04).Conclusions: Taken together, our results demonstrate that targeting the BCR signaling at multiple levels is a rational therapeutic strategy. Proapoptotic activity of FOXO1 is an important effector of SYK and AKT inhibition in DLBCLs and its expression is required for SYK-and AKT inhibitor-induced toxicity. The underlying mechanism linking FOXO1 activation and cell death involves caspase-dependent cleavage of transcriptional repressor DREAM and subsequent induction of a proapoptotic BCL2 family member, HRK. DisclosuresNo relevant conflicts of interest to declare.

Estrogen Receptor Beta Is a Novel Target in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous and aggressive malignant disease characterized by poor patient outcome and suboptimal front-line chemotherapy. To identify novel anti-AML compounds and potential molecular markers that confer drug-specificity, we performed a high-throughput screen of an in-house library consisting only of food-derived bioactive molecules (n=300). This screen was performed against the AML cell line (TEX), which has several properties of leukemia stem cells, the cells responsible for disease pathophysiology and patient relapse. This screen identified diosmetin as a novel anti-leukemia agent (IC50: 6.8 ± 1.7µm). Diosmetin (10µM) reduced clonogenic growth of primary AML patient cells with no effect on normal hematopoetic stem cells. In leukemia mouse xenografts, administration of diosmetin (50mg/kg) reduced tumor weight (>40%) without evidence of toxicity. Collectively, this shows that diosmetin is a novel anti-AML agent.Diosmetin-induced apoptosis (e.g., increased Annexin V staining and DNA fragmentation) was abolished in the presence of caspase-inhibitors. Importantly, diosmetin, which is a flavonoid and a known ROS-inducer, induced death in the presence of anti-oxidants demonstrating that death was not related to ROS activity. Instead, we demonstrate that diosmetin-induced caspase 8-, not caspase 9, mediated apoptosis that was facilitated by increased TNF-α.To identify diosmetin's molecular target we employed a novel method utilizing several free-online bioinformatics tools. Here, the protein data base (PBD), Pocket Similarity Search using Multi-Sketches (PoSSuM) and Database for Annotation, Visualization and Integrated Discovery (DAVID) tool, were used sequentially to identify potential diosmetin binding partners. This method identified the estrogen receptor (ER) as a potential molecular target for diosmetin's anti-leukemia activity.There are two types of ERs. When activated, ERα increases cell proliferation whereas ERβ inhibits proliferation. These opposite and opposing roles have led to the hypothesis that ER expression patterns dictate the cell's response to estrogen, as hormones like estradiol can bind to both ERα and ERβ with different affinities. To first determine to which ER diosmetin bound, we mapped the binding sites of both ERα and ERβ using the Discovery Studio (DS), Structure-Based-Design program and determined the relative affinity of diosmetin to these ERs. Diosmetin bound with far greater affinity to ERβ than ERα. Next, we interrogated publically available AML patient datasets to determine the expression pattern of ERα versus ERβ. Interestingly, we noted a subset of AML patients that upregulate ERβ but not ERα expression (log4 fold increase; p<0.001) suggesting the clinical relevance of targeting ERβ in a subset of AML patients.To assess the cell and molecular role of ERs, we measured ERα and ERβ levels in diosmetin sensitive and insensitive cell lines. Interestingly, diosmetin sensitive cell lines (TEX, LP1, AML2) display significantly elevated ER-β protein and mRNA levels (4 fold, as determined by Western blotting and qtPCR, respectively) compared to diosmetin insensitive cell lines (K562, DU145). This pattern was not observed for ER-α. Furthermore, this ER expression pattern was also observed in primary AML cells; as AML cells sensitive to diosmetin displayed an increase in ERβ, but not ERα, mRNA. Finally, ER reporter assays demonstrated that diosmetin binds and acts as a partial agonist in ERβ but not ERα reporter cells. Together, these results show that diosmetin binds to ERβ and that ERβ is functionally important to diosmetin's activity.In summary, these studies highlight ERβ as a potential novel therapeutic target for the treatment of AML. DisclosuresNo relevant conflicts of interest to declare.

Caco-2 cells infected with rotavirus release extracellular vesicles that express markers of apoptotic bodies and exosomes.

Previously, we showed that infecting human intestinal epithelial cells (Caco-2) with rotavirus (RV) increases the release of extracellular vesicles (EVs) with an immunomodulatory function that, upon concentration at 100,000×g, present buoyant densities on a sucrose gradient of between 1.10 to 1.18 g/ml (characteristic of exosomes) and higher than 1.24 g/ml (proposed for apoptotic bodies). The effect of cellular death induced by RV on the composition of these EV is unknown. Here, we evaluated exosome (CD63, Hsc70, and AChE) and apoptotic body (histone H3) markers in EVs isolated by differential centrifugation (4000×g, 10,000×g, and 100,000×g) or filtration/ultracentrifugation (100,000×g) protocols. When we infected cells in the presence of caspase inhibitors, Hsc70 and AChE diminished in EVs obtained at 100,000×g, but not in EVs obtained at 4000×g or 10,000×g. In addition, caspase inhibitors decreased CD63 and AChE in vesicles with low and high buoyant densities. Without caspase inhibitors, RV infection increased exosome markers in all of the EVs obtained by differential centrifugation. However, CD63 preferentially localized in the 100,000×g fraction and H3 only increased in EVs concentrated at 100,000×g and with high buoyant densities on a sucrose gradient. Thus, RV infection increases the release of EVs that, upon concentration at 100,000×g, are composed by exosomes and apoptotic bodies, which can partially be separated using sucrose gradients.

Centipedegrass extract induces apoptosis through the activation of caspases and the downregulation of PI3K/Akt and MAPK phosphorylation in leukemia cells.

Acute lymphoblastic leukemia(ALL), which involves the blood and bone marrow, is the most common type of cancer in children younger than 5years of age. Previous studies have investigated the effects of centipedegrass extract(CGE), which is mainly composed of maysin and its derivatives, and have demonstrated that it has various biological activities, including antioxidant and anti‑inflammatory activities, pancreatic lipase inhibitory activity, anti-adipogenic activity and insecticidal activity. To the best of our knowledge, this study is the first to investigate the anticancer effects of CGE in ALL cell lines and to elucidate the mechanisms underlying these effects. Cell viability was measured by thiazolyl blue tetrazolium blue(MTT) assay. Apoptosis, cell cycle progression and mitochondrial membrane potential(∆Ψm) were determined by flow cytometry. The effects of CGE on the phosphatidylinositol 3‑kinase(PI3K)/Akt pathway and mitogen‑activated protein kinases(MAPKs) were assessed by immunoblotting. PI3K, MAPK and caspase inhibitors were used to further confirm the molecular mechanisms involved. Our results clearly demonstrated that the proliferation of the ALL cells was significantly inhibited by CGE in a dose‑dependent manner. Apoptosis was accompanied by the induction of significant G1cell cycle arrest. The resulting alteration of the ∆Ψm increased the activity of caspase‑3/7. The induction of apoptosis was enhanced by the combined treatment of CGE with a PI3K inhibitor or an extracellular signal-regulated kinase (ERK) inhibitor, whereas the CGE‑induced apoptosis was inhibited in the presence of caspase inhibitors, such as z‑VAD‑fmk and z‑IETD‑fmk. Furthermore, CGE inhibited PI3K activity by decreasing the levels of phosphorylated (p‑)Akt, p‑BAD, and Bcl‑2 together with the levels of MAPKs, including p‑ERK and p‑JNK, but demonstrated no effects on p38MAPK. Thus, our data suggest that CGE may be a novel natural compound with potential for use as an antitumor agent in ALL.

Abstract 1342: RIP3 is downregulated in human myeloid leukemia cells and modulates apoptosis and caspase-mediated p65/RelA cleavage

Abstract Introduction. Impairment in cell death pathways represents a general characteristic of most cancer cells. The receptor-interacting protein kinase 3 (RIP3) associates with RIP1 in a necrosome complex that can induce necroptosis, apoptosis, or cell proliferation. The role of RIP3 in necroptosis and inflammation has been extensively studied, but its role in cancer remains poorly understood Methods. We analyzed the expression of RIP1 and RIP3 in CD34+ leukemia cells from a cohort of patients with acute myeloid leukemia (AML) and CD34+ cells from healthy donors. To analyze the potential advantages for myeloid malignant cells due to reduced RIP3 expression, we induced the expression of RIP3 in the DA1-3b mouse leukemia cell line. Results. RIP3 expression was significantly reduced in most AML samples, whereas the expression of RIP1 did not differ significantly. When re-expressed in the mouse DA1-3b leukemia cell line, RIP3 induced apoptosis, and necroptosis in the presence of caspase inhibitors. Surprisingly, the re-expression of a RIP3 mutant with an inactive kinase domain (RIP3-KD) induced significantly more and earlier apoptosis than wild-type RIP3 (RIP3 WT), indicating that the RIP3 kinase domain is an essential regulator of apoptosis/necroptosis in leukemia cells. The induced in vivo expression of RIP3-KD, but not RIP3 WT prolonged the survival of mice injected with leukemia cells. RIP3-KD-induced cell death but not RIP3 WT was significantly antagonized by an IKKβSSEE constitutively active mutant, showing that RIP3-KD-induced apoptosis, but not RIP3 WT-induced apoptosis, was dependent on NF-κB activity. The expression of RIP3-KD induced p65/RelA NF-κB subunit caspase-dependent cleavage, and a non-cleavable p65/RelA D361E mutant rescued cells from apoptosis. The protective effect of the p65/RelA D361E mutant against apoptosis was specific to RIP3-KD-induced cell death because no change in cell death was observed when apoptosis was instead induced by treatment with imatinib or DMSO. The p65/RelA D361E mutant was generated by mutating the INFD putative consensus recognition site for caspase-6. The caspase-6 inhibitor Z-VEID-fmk partially reduced the cell death induced by RIP3-KD and slightly reduced p65/RelA cleavage. p65/RelA cleavage appears to be at least partially mediated by caspase-6. Conclusions. These data indicate that RIP3 silencing in leukemia cells results in suppression of the complex regulation of the apoptosis/necroptosis switch and the modulation of the NF-κB pathway through the caspase-mediated cleavage of p65/RelA. Citation Format: Anne-Lucie Nugues, Hassiba Bouafia, Dominique Hetuin, Celine Berthon, Anne Loyens, Elisabeth Bertrand, Nathalie Jouy, Thierry Idziorek, Bruno Quesnel. RIP3 is downregulated in human myeloid leukemia cells and modulates apoptosis and caspase-mediated p65/RelA cleavage. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1342. doi:10.1158/1538-7445.AM2014-1342

RIP3 is downregulated in human myeloid leukemia cells and modulates apoptosis and caspase-mediated p65/RelA cleavage.

The receptor-interacting protein kinase 3 (RIP3) associates with RIP1 in a necrosome complex that can induce necroptosis, apoptosis, or cell proliferation. We analyzed the expression of RIP1 and RIP3 in CD34+ leukemia cells from a cohort of patients with acute myeloid leukemia (AML) and CD34+ cells from healthy donors. RIP3 expression was significantly reduced in most AML samples, whereas the expression of RIP1 did not differ significantly. When re-expressed in the mouse DA1-3b leukemia cell line, RIP3 induced apoptosis and necroptosis in the presence of caspase inhibitors. Transfection of RIP3 in the WEHI-3b leukemia cell line or in the mouse embryonic fibroblasts also resulted in increased cell death. Surprisingly, re-expression of a RIP3 mutant with an inactive kinase domain (RIP3-kinase dead (RIP3-KD)) induced significantly more and earlier apoptosis than wild-type RIP3 (RIP3-WT), indicating that the RIP3 kinase domain is an essential regulator of apoptosis/necroptosis in leukemia cells. The induced in vivo expression of RIP3-KD but not RIP3-WT prolonged the survival of mice injected with leukemia cells. The expression of RIP3-KD induced p65/RelA nuclear factor-κB (NF-κB) subunit caspase-dependent cleavage, and a non-cleavable p65/RelA D361E mutant rescued these cells from apoptosis. p65/RelA cleavage appears to be at least partially mediated by caspase-6. These data indicate that RIP3 silencing in leukemia cells results in suppression of the complex regulation of the apoptosis/necroptosis switch and NF-κB activity.

Anamorsin, a Novel Caspase-3 Substrate in Neurodegeneration

Activated caspases play a central role in the execution of apoptosis by cleaving endogenous substrates. Here, we developed a high throughput screening method to identify novel substrates for caspase-3 in a neuronal cell line. Critical steps in our strategy consist of two-dimensional electrophoresis-based protein separation and in vitro caspase-3 incubation of immobilized proteins to sort out direct substrates. Among 46 putative substrates identified in MN9D neuronal cells, we further evaluated whether caspase-3-mediated cleavage of anamorsin, a recently recognized cell death-defying factor in hematopoiesis, is a general feature of apoptosis. In vitro and cell-based cleavage assays indicated that anamorsin was specifically cleaved by caspase-3 but not by other caspases, generating 25- and 10-kDa fragments. Thus, in apoptosis of neuronal and non-neuronal cells induced by various stimuli including staurosporine, etoposide, or 6-hydroxydopamine, the cleavage of anamorsin was found to be blocked in the presence of caspase inhibitor. Among four tetrapeptide consensus DXXD motifs existing in anamorsin, we mapped a specific cleavage site for caspase-3 at DSVD(209)↓L. Intriguingly, the 25-kDa cleaved fragment of anamorsin was also detected in post-mortem brains of Alzheimer and Parkinson disease patients. Although the RNA interference-mediated knockdown of anamorsin rendered neuronal cells more vulnerable to staurosporine treatment, reintroduction of full-length anamorsin into an anamorsin knock-out stromal cell line made cells resistant to staurosporine-induced caspase activation, indicating the antiapoptotic function of anamorsin. Taken together, our approach seems to be effective to identify novel substrates for caspases and has the potential to provide meaningful insights into newly identified substrates involved in neurodegenerative processes.

Egr-1 Upregulates Siva-1 Expression and Induces Cardiac Fibroblast Apoptosis

The early growth response transcription factor Egr-1 controls cell specific responses to proliferation, differentiation and apoptosis. Expression of Egr-1 and downstream transcription is closely controlled and cell specific upregulation induced by processes such as hypoxia and ischemia has been previously linked to multiple aspects of cardiovascular injury. In this study, we showed constitutive expression of Egr-1 in cultured human ventricular cardiac fibroblasts, used adenoviral mediated gene transfer to study the effects of continuous Egr-1 overexpression and studied downstream transcription by Western blotting, immunohistochemistry and siRNA transfection. Apoptosis was assessed by fluorescence microscopy and flow cytometry in the presence of caspase inhibitors. Overexpression of Egr-1 directly induced apoptosis associated with caspase activation in human cardiac fibroblast cultures in vitro assessed by fluorescence microscopy and flow cytometry. Apoptotic induction was associated with a caspase activation associated loss of mitochondrial membrane potential and transient downstream transcriptional up-regulation of the pro-apoptotic gene product Siva-1. Suppression of Siva-1 induction by siRNA partially reversed Egr-1 mediated loss of cell viability. These findings suggest a previously unknown role for Egr-1 and transcriptional regulation of Siva-1 in the control of cardiac accessory cell death.

MLN4924, An Investigational Inhibitor Of NEDD8 Activating Enzyme (NAE), Is Active In Mantle Cell Lymphoma (MCL) Pre-Clinical Models and Enhances Rituximab Activity In Vivo

MCL is characterized by an aggressive clinical course and inevitable development of refractory disease despite early interventions that include immunotherapy (i.e. rituximab), multi-agent induction chemotherapy (that contains high-dose cytarabine or bortezomib) and often consolidation with high- dose chemotherapy and autologous stem cell support (HDC-ASCS) in first remission. The acquisition of resistant disease resulting in poor clinical outcomes stresses the need to develop alternative therapeutic strategies for MCL patients. To this end, we evaluated MLN4924, an NAE inhibitor which prevents the neddylation and activation of cullin-RING ubiquitin ligases, in MCL pre-clinical models. A panel of MCL cell lines and primary tumor cells isolated from MCL patients were exposed to escalating doses of MLN4924 alone or in the presence of caspase inhibitors. Changes in: apoptosis, cell cycle distribution, NFkB activity, protein or mRNA expression levels of Bcl-2 family members/cell cycle regulators were determined using the cell titer glo assay, flow cytometry, imagestream technology, and Western blotting, respectively. Subsequently, MCL cells were exposed to MLN4924 alone or in combination with various chemotherapy agents, and differences in cell viability were determined by measuring changes in ATP content. Standard 51Cr release assays were conducted to study the effects of MLN4924 on the anti-tumor activity of rituximab against MCL cell lines. For in vivo studies, severe combined immunodeficiency (SCID) mice were inoculated via tail vein injection (iv) with Granta cells (day 0) and assigned to observation, rituximab (at 10mg/kg/dose on days +2,+9,+16 and +23), cytarabine (at 20mg/kg/dose on days +3,+10,+17 and +24) or MLN4924 (at 180mg/kg/dose on days +1,+4,+8,+11,+15 and +18) with or without rituximab (same dose-schedule) or cytarabine (same dose-schedule). Differences in survival (measured as the time to development of limb paralysis) were evaluated by log-rank test across treatment arms. MLN4924 exposure resulted in a dose-, time-, and caspase-dependent cell death in the majority of the MCL cell lines and primary tumor cells tested (IC50=0.1-8.4µM). Of interest in MCL cell lines with lower IC50 (0.1-0.5µM), MLN4924 induced G1 phase cell cycle arrest, down-regulation of both Bcl-XL mRNA and protein levels, down-regulation of NFkB activity, and apoptosis (PARP cleavage). In addition, MLN4924 exhibited additive/synergistic effects when combined with chemotherapy agents (i.e. cytarabine, bortezomib or bendamustine). As a single agent, MLN4924 prolonged the survival of MCL-bearing SCID mice when compared to controls (median=56 vs 21 days; p<0.05). More importantly, MLN4924 in combination with rituximab (median survival not reached at 90 days) led to an improved survival compared to rituximab (median survival 40 days) or MLN4924 alone (56 days) (P=0.045). Our data suggest that MLN4924 has significant activity in MCL pre-clinical models, possibly related to effects on NF-κB activity and Bcl-XL down-regulation, and can potentiate rituximab activity in vivo. These findings support further investigation of MLN4924 +/- rituximab in the treatment of MCL. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund) Disclosures:Czuczman:Genetech, Onyx, Celgene, Astellas, Millennium, Mundipharma: Advisory Committees Other.

Deltamethrin induced an apoptogenic signalling pathway in murine thymocytes : exploring the molecular mechanism

Deltamethrin (DLM) is a well-known pyrethroid insecticide; however, the immunotoxic effects of DLM on the mammalian system and its mechanism is still unclear. This study has been designed to first observe the binding affinity of DLM to immune cell receptors and its effects on the immune system. The docking score revealed that DLM has a strong binding affinity towards the CD4 and CD8 receptors. DLM induces apoptosis in murine thymocytes in a concentration-dependent manner. The ear\ly markers of apoptosis such as enhanced reactive oxygen species (ROS) and caspase-3 activation are evident as early as 1 h by 25 and 50 μM DLM. Glutathione (GSH) depletion has also been observed at 1 h by 50 μM DLM concentration. In cell-cycle studies using flow cytometry, the fraction of hypodiploid cells has gradually increased with all the concentrations of DLM at 18 h. The Annexin V binding assay measures the effect of DLM on apoptotic and necrotic cells. The apoptotic cells raised from 18.6% to 35.21% (10-50 μM DLM) at 18 h. N-acetyl cysteine (NAC) effectively reduced the percentage of apoptotic cells which is increased by DLM. In contrast, buthionine sulfoximine (BSO) caused an elevation in the percentage of apoptotic cells. These results demonstrate that caspase activation, ROS activation and GSH act as critical mediators in a DLM-induced apoptogenic signalling pathway in murine thymocytes. In the presence of caspase inhibitor, the percentage of apoptotic cells is partially decreased. Thus, there may be the possibility of some other caspase-independent pathways in DLM-induced apoptosis.

Cell death of L-929 cells induced by cytotoxic complex Tag7-Hsp70 is analogous to the death of the same cells induced by TNF-α

The identification and studying the molecular bases of functioning of new cytotoxic agents finds an important implication in developing drugs for fighting with tumors. While investigating the cytotoxic action of protein complex Tag7-Hsp70 which was opened in our laboratory previously we found that Tag7-Hsp70 demonstrated the same specificity in regard to different tumor target cells as it was for classical cytokine TNF-α. L-929 cells and Jurkat cells appeared to be good targets representing up to 30% of dead cells within a population and HeLa cells--bad targets representing less than 5% of dead cells after 20 h of incubation with either of the cytotoxic agents. While investigating the action of either TNF-α or Tag7-Hsp70 on L-929 cells we detected two peaks of death: after 3 h and after 20 h. For both cytotoxic agents we observed the first, smaller (13-15%), peak to be eliminated after the addition of caspase inhibitor YVAD-CHO and the second, greater (25-30%), peak to become even bigger in presence of caspase inhibitor. Probably, protein complex Tag7-Hsp70 interacts like TNF-α with a receptor on the surface of tumor cells that results in triggering two alternative mechanisms of programmed cell death: apoptosis and necroptosis.

Pea lectin inhibits growth of Ehrlich ascites carcinoma cells by inducing apoptosis and G2/M cell cycle arrest in vivo in mice

Pea (Pisum sativum L.) lectin is known to have interesting pharmacological activities and of great interest on biomedical research. In the current research pea lectin was purified followed by ion exchange chromatography on DEAE column and affinity chromatography on glucose-sepharose column. The lectin shown 11.7–84% inhibitory effect against Ehrlich ascites carcinoma (EAC) cells at the concentration range of 8–120μg/ml in RPMI 1640 medium as determined by MTT assay. Pea lectin was also shown 63% and 44% growth inhibition against EAC cells in vivo in mice when administered 2.8mg/kg/day and 1.4mg/kg/day (i.p.) respectively for five consequent days. When Pea lectin injected into the EAC bearing mice for 10 days its significantly increased the hemoglobin and RBC with the decreased of WBC levels toward the normal. Apoptotic cell morphological change of the treated EAC cells of mice was determined by fluorescence and optical microscope. Interestingly, cell growth inhibition of the lectin was significantly reduced in the presence of caspase inhibitors. Treatment with the lectin caused the cell cycle arrest at G2/M phase of EAC cells which was determined by flow cytometry. The expression of apoptosis-related genes, Bcl-2, Bcl-X and Bax was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). Intensive increase of Bax gene expression and totally despaired of Bcl-2 and Bcl-X gene expression were observed in the cells treated with Pea lectin for five consecutive days.

A component of gamma-radiation-induced cell death in E. coli is programmed and interlinked with activation of caspase-3 and SOS response

The current study deals with the molecular mechanism of radiation-induced cell death (RICD) in Escherichia coli. Irradiated E. coli cells displayed markers similar to those found in eukaryotic programmed cell death (PCD) such as caspase-3 activation and phosphatidylserine externalization. RICD was found to be suppressed upon pretreatment with sublethal concentrations of rifampicin or chloramphenicol, indicating the requirement of de novo gene expression. RICD was also found to be inhibited by cell permeable inhibitors of caspase-3 or poly (ADP-ribose) polymerase, indicating the involvement of PCD during RICD in E. coli. Radiation-induced SOS response was alleviated as observed with decrease in LexA level and also reduced cell filamentation frequency in the presence of caspase inhibitor. Further, the inhibitor-mediated rescue was not observed in single-gene knockouts of umuC, umuD, recB and ruvA, the genes which are associated with SOS response. This implies a linkage between SOS response and PCD in radiation-exposed E. coli cells.