Death Receptor-mediated Apoptosis Research Articles

Functional Consequences for Apoptosis by Transcription Elongation Regulator 1 (TCERG1)-Mediated Bcl-x and Fas/CD95 Alternative Splicing.

Here, we present evidence for a specific role of the splicing-related factor TCERG1 in regulating apoptosis in live cells by modulating the alternative splicing of the apoptotic genes Bcl-x and Fas. We show that TCERG1 modulates Bcl-x alternative splicing during apoptosis and its activity in Bcl-x alternative splicing correlates with the induction of apoptosis, as determined by assessing dead cells, sub-G1-phase cells, annexin-V binding, cell viability, and cleavage of caspase-3 and PARP-1. Furthermore, the effect of TCERG1 on apoptosis involved changes in mitochondrial membrane permeabilization. We also found that depletion of TCERG1 reduces the expression of the activated form of the pro-apoptotic mitochondrial membrane protein Bak, which remains inactive by heterodimerizing with Bcl-xL, preventing the initial step of cytochrome c release in Bak-mediated mitochondrial apoptosis. In addition, we provide evidence that TCERG1 also participates in the death receptor-mediated apoptosis pathway. Interestingly, TCERG1 also modulates Fas/CD95 alternative splicing. We propose that TCERG1 sensitizes a cell to apoptotic agents, thus promoting apoptosis by regulating the alternative splicing of both the Bcl-x and Fas/CD95 genes. Our findings may provide a new link between the control of alternative splicing and the molecular events leading to apoptosis.

Abstract B72: Environmental temperature-induced chronic stress drives therapeutic resistance in murine tumor models through β2-adrenergic receptor activation

Abstract Pre-clinical evaluation of novel therapies primarily relies on mouse models for predicting efficacy and toxicity. Unfortunately, when taken into the clinic, promising therapies often fail to achieve expected results in patients or unexpected toxicities are encountered. Emerging evidence now suggests that laboratory housing conditions, including ad libitum feeding and limited physical activity, can lead to metabolic abnormalities which in turn directly affect experimental outcomes in models of diseases such as obesity. We are particularly interested in the ramifications of housing temperature in tumor-bearing mice. IACUC guidelines dictate that all animal facilities must select a single temperature within the range of 20-26˚C (standard temperature, ST) to maintain mice, even though the thermoneutral temperature of mice ranges from 29-31˚C (TT). Because mice are able to maintain a normal body temperature of 37˚C, investigators have not been concerned, but in fact mice must generate a significant amount of heat through adaptive thermogenesis, and this is a norepinephrine (NE) driven process. The tumor promoting effects of catecholamines (epinephrine and norepinephrine) are currently under investigation in several labs. We have previously reported that at ST, the anti-tumor immune response is severely suppressed and when cold stress is alleviated by housing at TT, significantly higher numbers of tumor specific CD8 T-cells and fewer suppressor cells (MDSC, Tregs) are seen, correlating with significantly improved tumor control. Now we have observed that housing mice at TT instead of ST also significantly improves the response of pancreatic tumors to apoptosis inducing therapies in both immunocompetant and SCID mouse models. We found that a syngeneic tumor, Pan02, in C57BL/6 mice showed increased sensitivity to cisplatin when mice were placed at 30˚C vs. 22˚C. Similarly, human cell lines (MiaPaca2, BxPC3) and patient derived xenografts also demonstrated improved responses to both cisplatin and a second therapy, Apo2L/TRAIL, when mice were placed at thermoneutrality. Examination of the tumors revealed that the norepinephrine levels and the numbers of tyrosine hydroxylase-positive macrophages, a major source of norepinephrine, decreased in the tumors of mice housed at TT compared with those at ST. Thus, we hypothesized that tumor cells respond to the elevated levels of norepinephrine in the microenvironment in chronically cold stressed animals by increasing the expression of key survival molecules. Subsequent analysis of tumors from mice housed at TT revealed that the expression of anti-apoptotic molecules Bcl-2, Bcl-XL, Mcl-1 and phosphorylated BAD112 were decreased compared to those housed at ST. Tumor cells were found to express β1,2 adrenergic receptors and in vitro treatment demonstrated that adrenergic signaling can directly induce the expression of Bcl-2 and Bcl-XL in a time dependent manner in human pancreatic tumor cell lines, and that this increased protein expression correlates with increased resistance to both chemotherapeutic and death receptor-mediated apoptosis. In vivo, treatment of tumor-bearing mice with the β1, 2-blocker, propranolol, increased the response of tumors in mice housed at 22˚C to Apo2L/TRAIL, but not mice housed at 30˚C. Resistance to apoptotic therapy at ST was lost by shRNA knock down of β2-AR (MiaPaca2). Altogether, these findings have important implications for the study of immunotherapies which depend on both activation of the anti-tumor immune response and the sensitivity of tumors to induction of apoptosis. Our results support the idea that adrenergic signaling in the tumor microenvironment (at ST) can greatly impact the testing of novel therapies and suggests strategies for improving their efficacy both in mouse models and in the clinic where patients experience a variety of stresses. Supported by the Roswell Park Alliance Foundation and NIH R01 CA135368. Citation Format: Jason W-L Eng, Chelsey B. Reed, Kathleen Kokolus, Mark Bucsek, Rose Pitoniak, Adam T. Utley, WenWee Ma, Elizabeth A. Repasky, Bonnie L. Hylander. Environmental temperature-induced chronic stress drives therapeutic resistance in murine tumor models through β2-adrenergic receptor activation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B72.

TGR5 is essential for bile acid-dependent cholangiocyte proliferation in vivo and in vitro

ObjectiveCholestatic liver diseases in humans as well as bile acid (BA)-feeding and common bile duct ligation (CBDL) in rodents trigger hyperplasia of cholangiocytes within the portal fields. Furthermore, elevation of...

Gene Expression Ratios Lead to Accurate and Translatable Predictors of DR5 Agonism across Multiple Tumor Lineages.

Death Receptor 5 (DR5) agonists demonstrate anti-tumor activity in preclinical models but have yet to demonstrate robust clinical responses. A key limitation may be the lack of patient selection strategies to identify those most likely to respond to treatment. To overcome this limitation, we screened a DR5 agonist Nanobody across >600 cell lines representing 21 tumor lineages and assessed molecular features associated with response. High expression of DR5 and Casp8 were significantly associated with sensitivity, but their expression thresholds were difficult to translate due to low dynamic ranges. To address the translational challenge of establishing thresholds of gene expression, we developed a classifier based on ratios of genes that predicted response across lineages. The ratio classifier outperformed the DR5+Casp8 classifier, as well as standard approaches for feature selection and classification using genes, instead of ratios. This classifier was independently validated using 11 primary patient-derived pancreatic xenograft models showing perfect predictions as well as a striking linearity between prediction probability and anti-tumor response. A network analysis of the genes in the ratio classifier captured important biological relationships mediating drug response, specifically identifying key positive and negative regulators of DR5 mediated apoptosis, including DR5, CASP8, BID, cFLIP, XIAP and PEA15. Importantly, the ratio classifier shows translatability across gene expression platforms (from Affymetrix microarrays to RNA-seq) and across model systems (in vitro to in vivo). Our approach of using gene expression ratios presents a robust and novel method for constructing translatable biomarkers of compound response, which can also probe the underlying biology of treatment response.

Cytotoxicity of atropine to human corneal epithelial cells by inducing cell cycle arrest and mitochondrion-dependent apoptosis

Atropine is an anticholinergic drug for mydriasis in eye clinic, and its abuse might be cytotoxic to the cornea and result in blurred vision. However, the cytotoxicity of atropine to the cornea and its cellular and molecular mechanisms remain unknown. In this study, we investigated the cytotoxicity of atropine to corneal epithelium and its underlying mechanisms using an in vitro model of non-transfected human corneal epithelial (HCEP) cells. Our results showed that atropine, above the concentration of 0.3125g/l (1/32 of its therapeutic dosage in eye clinic), had a dose- and time-dependent toxicity to HCEP cells by inducing morphological abnormality, cytopathic effect, viability decline, and proliferation retardation. Moreover, the proliferation-retarding effect of atropine on the cells was achieved by inducing G1/S phase arrest and downregulation of E-cadherin and β-catenin. Besides, atropine also had an apoptosis-inducing effect on the cells by inducing phosphatidylserine externalization, plasma membrane permeability elevation, DNA fragmentation and apoptotic body formation. Furthermore, atropine could also induce activations of caspase-2, -3 and -9, disruption of mitochondrial transmembrane potential, downregulation of Bcl-2 and Bcl-xL, upregulation of Bax and Bad, and upregulation of cytoplasmic cytochrome c and apoptosis-inducing factor, implying a death receptor-mediated mitochondrion-dependent pathway is most probably involved in the apoptosis of HCEP cells induced by atropine. Taken together, our results suggest that atropine has remarkable cytotoxicity to HCEP cells by inducing cell cycle arrest and death receptor-mediated mitochondrion-dependent apoptosis.

Conservation of structure and function in vertebrate c-FLIP proteins despite rapid evolutionary change

Cellular FLICE-like inhibitory protein (c-FLIP, gene symbol CFLAR) was first identified as a negative regulator of death receptor-mediated apoptosis in mammals. To understand the ubiquity and diversity of the c-FLIP protein subfamily during evolution, c-FLIP orthologs were identified from a comprehensive range of vertebrates, including birds, amphibians, and fish, and were characterized by combining experimental and computational analysis. Predictions of three-dimensional protein structures and molecular phylogenetic analysis indicated that the conserved structural features of c-FLIP proteins are all derived from an ancestral caspase-8, although they rapidly diverged from the subfamily consisting of caspases-8, -10, and -18. The functional role of the c-FLIP subfamily members is nearly ubiquitous throughout vertebrates. Exogenous expression of non-mammalian c-FLIP proteins in cultured mammalian cells suppressed death receptor-mediated apoptosis, implying that all of these proteins possess anti-apoptotic activity. Furthermore, non-mammalian c-FLIP proteins induced NF-κB activation much like their mammalian counterparts. The CFLAR mRNAs were synthesized during frog and fish embryogenesis. Overexpression of a truncated mutant of c-FLIP in the Xenopus laevis embryos by mRNA microinjection caused thorax edema and abnormal constriction of the abdomen. Depletion of cflar transcripts in zebrafish resulted in developmental abnormalities accompanied by edema and irregular red blood cell flow. Thus, our results demonstrate that c-FLIP/CFLAR is conserved in both protein structure and function in several vertebrate species, and suggest a significant role of c-FLIP in embryonic development.

Modulation of PKC signaling and induction of apoptosis through suppression of reactive oxygen species and tumor necrosis factor receptor 1 (TNFR1): key role of quercetin in cancer prevention.

Cancer cells are characterized by increased production of reactive oxygen species (ROS) and an altered redox environment as compared to normal cells. Continuous accumulation of ROS triggers oxidative stress leading to hyper-activation of signaling pathways that promote cell proliferation, survival, and metabolic adaptation to the tumor microenvironment. Therefore, antioxidants are proposed to contribute to cancer prevention. Protein kinase C (PKC) is a crucial regulator of diverse cellular processes and contributes to cancer progression. The activation of PKC is partially dependent on ROS signaling. In the present study, cancer preventive activity of natural flavonoid quercetin is analyzed in ascite cells of Dalton's lymphoma-bearing mice. The total ROS level and activity of PKC were downregulated after quercetin treatment in lymphoma-bearing mice. Quercetin modulates the expression of almost all isozymes of classical, novel, and atypical PKC as well as downregulates the level and expression of PKCα. Further, quercetin improves apoptotic potential, as observed by the levels of caspase 3, caspase 9, PARP, PKCδ, and nuclear condensation. Additionally, quercetin reduces cell survival and promotes death receptor-mediated apoptosis via differential localization of the TNFR1 level in ascite cells. The overall result suggests the cancer preventive activity of quercetin via the induction of apoptosis and modulates PKC signaling with the reduction of oxidative stress in ascite cells of lymphoma-bearing mice.

Nortriptyline induces mitochondria and death receptor-mediated apoptosis in bladder cancer cells and inhibits bladder tumor growth in vivo

Nortriptyline (NTP), an antidepressant, has antitumor effects on some human cancer cells, but its effect on human bladder cancer cells is not known. In this study, we used a cell viability assay to demonstrate that NTP is cytotoxic to human TCCSUP and mouse MBT-2 bladder cancer cells in a concentration and time-dependent manner. We also performed cell cycle analysis, annexin V and mitochondrial membrane potential assays, and Western blot analysis to show that NTP inhibits cell growth in these cells by inducing both mitochondria-mediated and death receptor-mediated apoptosis. Specifically, NTP increases the expression of Fas, FasL, FADD, Bax, Bak, and cleaved forms of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. In addition, NTP decreases the expression of Bcl-2, Bcl-xL, BH3 interacting domain death agonist, X-linked inhibitor of apoptosis protein, and survivin. Furthermore, NTP-induced apoptosis is associated with reactive oxygen species (ROS) production, which can be reduced by antioxidants, such as N-acetyl-L-cysteine. Finally, we showed that NTP suppresses tumor growth in mice inoculated with MBT-2 cells. Collectively, our results suggest that NTP induces both intrinsic and extrinsic apoptosis in human and mouse bladder cancer cells and that it may be a clinically useful chemotherapeutic agent for bladder cancer in humans.

Deletion of FADD in macrophages and granulocytes results in RIP3- and MyD88-dependent systemic inflammation.

Myeloid cells, which include monocytes, macrophages, and granulocytes, are important innate immune cells, but the mechanism and downstream effect of their cell death on the immune system is not completely clear. Necroptosis is an alternate form of cell death that can be triggered when death receptor-mediated apoptosis is blocked, for example, in stimulated Fas-associated Death Domain (FADD) deficient cells. We report here that mice deficient for FADD in myeloid cells (mFADD-/-) exhibit systemic inflammation with elevated inflammatory cytokines and increased levels of myeloid and B cell populations while their dendritic and T cell numbers are normal. These phenotypes were abolished when RIP3 deficiency was introduced, suggesting that systemic inflammation is caused by RIP3-dependent necroptotic and/or inflammatory activity. We further found that loss of MyD88 can rescue the systemic inflammation observed in these mice. These phenotypes are surprisingly similar to that of dendritic cell (DC)-specific FADD deficient mice with the exception that DC numbers are normal in mFADD-/- mice. Together these data support the notion that innate immune cells are constantly being stimulated through the MyD88-dependent pathway and aberrations in their cell death machinery can result in systemic effects on the immune system.

Caspase 8 in some dermatoses

Caspase 8 is closely associated with death receptor-mediated apoptosis. In recent years, some progress has been made in studies on the roles of caspase 8 in apoptosis. Besides initiating apoptosis, caspase 8 has many other biological functions, such as participating in T cell proliferation and immune responses, cell adhesion/migration, vascular endothelium formation, etc. As a key initiator of the Fas-mediated apoptotic pathway, caspase 8 is closely associated with repair of skin damage, cicatrization and lichen planus, and also plays some roles in counteracting inflammation. Key words: Skin diseases; Caspase 8; Apoptosis

Essential oil of Cephalotaxus griffithii needle inhibits proliferation and migration of human cervical cancer cells: involvement of mitochondria-initiated and death receptor-mediated apoptosis pathways

This study was conducted to determine the effect of Cephalotaxus griffithii needle essential oil (CGNO) on proliferation and migration of human cervical cancer (HCC) cells. CGNO treatment decreased the viability of all the tested HCC (HeLa, ME-180 and SiHa) cells. Morphological and DNA fragmentation analysis of CGNO-treated HeLa cells indicated the involvement of apoptosis in inducing HCC cell death. CGNO increased mitochondrial membrane depolarisation and upregulated the expression of caspase-9, caspase-8, caspase-3 and cleaved-PARP. The activity of caspase-8 and caspase-9 was also significantly increased. Wound healing and transwell migration assay demonstrated that CGNO significantly inhibited the migration of HeLa cells to close a scratched wound and also inhibited their migration through filter towards a chemotactic stimulus. Taken together, these results indicated that CGNO inhibited the proliferation and migration of HCC cells. Of note, CGNO induced HeLa cell death through mitochondria-initiated and death receptor-mediated apoptosis pathway.

Mitochondrial division inhibitor 1 (mdivi-1) enhances death receptor-mediated apoptosis in human ovarian cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) based strategy is a promising targeted therapeutic approach for the treatment of a variety of cancers including ovarian cancer. However, the inherent or acquired resistance of tumor cells to TRAIL limits the potential application of TRAIL-mediated therapy. In this study, we identified that mitochondrial division inhibitor-1 (mdivi-1) is able to enhance the sensitivity of human ovarian cancer cells to death receptor ligands including TRAIL, FAS ligands, and TNF-α. Importantly, the combination of TRAIL and mdivi-1 has no apparent cytotoxic effect on non-transformed human cells, indicating a significant therapeutic window. We identified that caspase-8 and not the modulation of TRAIL receptors is required for the combination effect of TRAIL and mdivi-1. We further demonstrated that the enhanced efficacy of combination of mdivi-1 and death ligands is not dependent on the originally reported target of mdivi-1, Drp1, and is also not dependent on the two important pro-apoptotic Bcl-2 family proteins Bax and Bak. Thus, our study presents a novel strategy in enhancing the apoptotic effect of death receptor ligands and provides a new effective TRAIL-based combination approach for treating human ovarian cancer.

Autoreactive T cells induce necrosis and not BCL-2-regulated or death receptor-mediated apoptosis or RIPK3-dependent necroptosis of transplanted islets in a mouse model of type 1 diabetes.

Type 1 diabetes results from T cell-mediated destruction of pancreatic beta cells. The mechanisms of beta cell destruction in vivo, however, remain unclear. We aimed to test the relative roles of the main cell death pathways: apoptosis, necrosis and necroptosis, in beta cell death in the development of CD4(+) T cell-mediated autoimmune diabetes. We altered expression levels of critical cell death proteins in mouse islets and tested their ability to survive CD4(+) T cell-mediated attack using an in vivo graft model. Loss of the B cell leukaemia/lymphoma 2 (BCL-2) homology domain 3-only proteins BIM, PUMA or BID did not protect beta cells from this death. Overexpression of the anti-apoptotic protein BCL-2 or combined deficiency of the pro-apoptotic multi-BCL2 homology domain proteins BAX and BAK also failed to prevent beta cell destruction. Furthermore, loss of function of the death receptor Fas or its essential downstream signalling molecule Fas-associated death domain (FADD) in islets was also not protective. Using electron microscopy we observed that dying beta cells showed features of necrosis. However, islets deficient in receptor-interacting serine/threonine protein kinase 3 (RIPK3), a critical initiator of necroptosis, were still normally susceptible to CD4(+) T cell-mediated destruction. Remarkably, simultaneous inhibition of apoptosis and necroptosis by combining loss of RIPK3 and overexpression of BCL-2 in islets did not protect them against immune attack either. Collectively, our data indicate that beta cells die by necrosis in autoimmune diabetes and that the programmed cell death pathways apoptosis and necroptosis are both dispensable for this process.

Abstract 799: Diminishing Mcl-1 protein leads to apoptosis of acute myeloid leukemia cells responding to all trans retinoic acid differentiation

Abstract All trans retinoic acid (ATRA) is successfully used for the treatment of acute promyelocytic leukemia (APL) through inducing terminal differentiation and death receptor-mediated apoptosis. However, ATRA has limited clinic efficacy in non-APL acute myeloid leukemia (AML) patients. To extend ATRA therapy to AML (non-APL), cell death and differentiation induction were tested and compared in a panel of AML cell lines after treatment with ATRA for 2, 4 and 6 days based on the staining of CD11b and annexin V, respectively. ATRA treatment in APL NB4 cells induces differentiation but not apoptosis at 2 days. ATRA differentiation peaks at 4 days and apoptosis becomes measurable at 4 days and peaks at 6 days. Western blot analysis revealed that both TRAIL and DR5 were induced by ATRA at 4 days, supporting that a death-receptor-mediated apoptosis program is initiated by ATRA. The mitochondrial apoptotic program was determined by measuring the levels of Bcl-2, Mcl-1 and the cleaved caspase-9. Although Bcl-2 protein was depleted, Mcl-1 protein was significantly increased after treatment with ATRA at 2 days. As the treatment continued to 4 and 6 days, the Mcl-1 protein levels declined and cleaved caspase 9 was detected. Similar phenomena were observed in non-APL HL-60 cells responding to ATRA-induced differentiation, but not in ATRA differentiation resistant NB4 and HL-60 subclones. Silencing of Mcl-1 with siRNA sensitized ATRA-induced apoptosis with decreased differentiation in NB4 cells treated with ATRA for 2 days. HL-60 cells with overexpression of Mcl-1 increased ATRA-induced differentiation with diminished apoptosis after treatment with ATRA for 6 days. Mechanistic studies of Mcl-1 induction by ATRA revealed that both Akt/mTOR and MEK/ERK signaling were activated associated with increased phosphorylation of glycogen synthase kinase 3β(GSK3β) and ribosomal S6 protein. Since inactivation of GSK3β by phosphorylation stabilizes Mcl-1 protein and S6 phosphorylation increases Mcl-1 protein translation, ATRA increases Mcl-1 levels through increasing translation and stability. Akt inhibitor Ly294002 and MEK inhibitor U0126, but not mTOR inhibitor rapamycin, blocked Mcl-1 induction and converted ATRA into an apoptosis inducer in HL-60 cells. More intriguingly sorafenib, a multi-kinase inhibitor, antagonized ATRA induction of Mcl-1, p-GSK3β and p-S6, but not Bcl-2 reduction, and synergistically induced apoptosis with ATRA in a panel of non-APL cell lines and primary AML cells. Our data suggest that Mcl-1 protein levels contribute to balance between cell death and differentiation of AML cells treated with ATRA and that ATRA therapy could be extended to non-APL cells by augmented cell death induction by diminishing Mcl-1 protein. Note: This abstract was not presented at the meeting. Citation Format: Rui Wang, Lijuan Xia, Janice Gabrilove, Samuel Waxman, Yongkui Jing. Diminishing Mcl-1 protein leads to apoptosis of acute myeloid leukemia cells responding to all trans retinoic acid differentiation. [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 799. doi:10.1158/1538-7445.AM2014-799

Abstract 2282: Calmodulin binding to DR-5 and the role of CaM-DR-5 binding in DR-5-mediated DISC formation in breast cancer

Abstract Molecular classification of breast cancers into receptor-based subtypes, including estrogen positive or triple negative breast cancers, establish targets that guide individualized treatment for patients (Journal of clinical oncology:27, 1153-1154, 2009). However, drug resistance observed for breast cancer raises the challegens for the breast cancer treatment (CA: a cancer journal for clinicians 59, 303-313, 2009). Targeting the death receptor-5 (DR-5) to induce breast cancer apoptosis is a promising strategy for breast cancer treatment (Immunity 3, 673-682, 1995, Breast Cancer Res Treat 113, 217-230, 2009). TRA-8 is a DR-5 specific agonistic antibody (Nature medicine 7, 954-960, 2001), and TRA-8 can induce apoptosis in breast cancer cells (Clinical cancer research: 9, 3731-3741, 2003). TRA-8 signal DR-5-mediated apoptotic signal via formation of the death inducing signaling complex (DISC) at the DR-5 death domain (Immunity 12, 599-609, 2000). Calmodulin (CaM) is a mediator of calcium signaling and has been shown to play a role in regulating DR-5 mediated apoptosis (Biochem Cell Biol 87, 919-926, 2009). Understanding the mechanism of DR-5-CaM interaction and the role of CaM-DR-5 binding in regulating DR-5 mediated DISC formation, may unveil CaM-DR-5 binding as a potential target to modulate DR-5 mediated apoptosis. In this study we characterize an interaction between CaM and DR-5 and the role of CaM-DR-5 binding in DR-5-mediated DISC formation in MCF-7 and MDA-MB-231 breast cancers. Complex-immunoprecipitation of DR-5 from MCF-7 and MDA-MB-231 lysates demonstrated endogenous CaM and DR-5 form a complex. Native PAGE of recombinant 6xHis-SUMO-CaM incubated with MCF-7 and MDA-MB-231 lysates showed the formation of a CaM and DR-5 complex. CaM pull down of DR-5 from MCF-7 and MDA-MB231 lysates in the presence of 1 mM Ca2+ or 2 mM EGTA showed CaM/DR-5 interaction is calcium dependent. TRA-8 activation of DR-5 resulted in CaM recruitment into the DR-5 mediated DISC. Additionally, Either EGTA or CaM antagonist trifluopherazine (TFP) attenuated DR-5 mediated DISC formation in MCF-7 and MDA-MB-231 breast cancers. We present evidence for the Ca2+ dependent CaM and DR-5 interaction and a role of CaM-DR-5 binding in regulating DR-5 mediated DISC formation in breast cancer cells. Results from this study provide the basis for the further characterization of CaM-DR5 interactions and its important role in cellular activities, which could lead to novel strategies for effectively regulating DR-5-mediated apoptosis in breast cancer. Citation Format: Romone M. Fancy, Hong Wang, Tong Zhou, Yuhua Song. Calmodulin binding to DR-5 and the role of CaM-DR-5 binding in DR-5-mediated DISC formation in breast cancer. [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 2282. doi:10.1158/1538-7445.AM2014-2282

Involvement of the PI3K and ERK signaling pathways in largemouth bass virus-induced apoptosis and viral replication

Increased reports demonstrated that largemouth Bass, Micropterus salmoides in natural and artificial environments were always suffered from an emerging iridovirus disease, largemouth Bass virus (LMBV). However, the underlying mechanism of LMBV pathogenesis remained largely unknown. Here, we investigated the cell signaling events involved in virus induced cell death and viral replication in vitro. We found that LMBV infection in epithelioma papulosum cyprini (EPC) cells induced typical apoptosis, evidenced by the appearance of apoptotic bodies, cytochrome c release, mitochondrial membrane permeabilization (MMP) destruction and reactive oxygen species (ROS) generation. Two initiators of apoptosis, caspase-8 and caspase-9, and the executioner of apoptosis, caspase-3, were all significantly activated with the infection time, suggested that not only mitochondrion-mediated, but also death receptor-mediated apoptosis were involved in LMBV infection. Reporter gene assay showed that the promoter activity of transcription factors including p53, NF-κB, AP-1 and cAMP response element-binding protein (CREB) were decreased during LMBV infection. After treatment with different signaling pathway inhibitors, virus production were significantly suppressed by the inhibition of phosphatidylinositol 3-kinase (PI3K) pathway and extracellular-signal-regulated kinases (ERK) signaling pathway. Furthermore, LMBV infection induced apoptosis was enhanced by PI3K inhibitor LY294002, but decreased by addition of ERK inhibitor UO126. Therefore, we speculated that apoptosis was sophisticatedly regulated by a series of cell signaling events for efficient virus propagation. Taken together, our results provided new insights into the molecular mechanism of ranavirus infection.

The physiological relevance of death receptor-mediated apoptosis.

Andreas Strasser describes the first paper to outline a physiological role for apoptosis downstream of the death receptor FAS in lpr mice.

Poly(ADP-ribose) Polymerase Inhibitors Sensitize Cancer Cells to Death Receptor-mediated Apoptosis by Enhancing Death Receptor Expression

Recombinant human tumor necrosis factor-α-related apoptosis inducing ligand (TRAIL), agonistic monoclonal antibodies to TRAIL receptors, and small molecule TRAIL receptor agonists are in various stages of preclinical and early phase clinical testing as potential anticancer drugs. Accordingly, there is substantial interest in understanding factors that affect sensitivity to these agents. In the present study we observed that the poly(ADP-ribose) polymerase (PARP) inhibitors olaparib and veliparib sensitize the myeloid leukemia cell lines ML-1 and K562, the ovarian cancer line PEO1, non-small cell lung cancer line A549, and a majority of clinical AML isolates, but not normal marrow, to TRAIL. Further analysis demonstrated that PARP inhibitor treatment results in activation of the FAS and TNFRSF10B (death receptor 5 (DR5)) promoters, increased Fas and DR5 mRNA, and elevated cell surface expression of these receptors in sensitized cells. Chromatin immunoprecipitation demonstrated enhanced binding of the transcription factor Sp1 to the TNFRSF10B promoter in the presence of PARP inhibitor. Knockdown of PARP1 or PARP2 (but not PARP3 and PARP4) not only increased expression of Fas and DR5 at the mRNA and protein level, but also recapitulated the sensitizing effects of the PARP inhibition. Conversely, Sp1 knockdown diminished the PARP inhibitor effects. In view of the fact that TRAIL is part of the armamentarium of natural killer cells, these observations identify a new facet of PARP inhibitor action while simultaneously providing the mechanistic underpinnings of a novel therapeutic combination that warrants further investigation.

Retracted Article: Interference of Mucin 1 inhibits the growth of liver cancer cells by inducing mitochondria-mediated and death receptor-mediated cell apoptosis.

This article has been retracted due to the authors wish to retract this article “Interference of Mucin1 inhibits the growth of liver cancer cells by inducing mitochondria mediated and death receptor mediated cell apoptosis” by Yongzhen Zhai, Deping Ding, LiyaWei, Enjing Chen, Guohe Feng, published in Tumor Biology, 2014, DOI: 10.1007/s13277-014-2120-9. Concerns were raised on the accuracy of the data presented and as consequence in the results and conclusions of the article for which the authors accept full responsibility. The authors apologize to the readers, reviewers, and editors for publishing this erroneous data.

C-FLIP links mTORC2 to apoptosis.

c-FLIP links mTORC2 to apoptosis.