Proapoptotic Stimuli Research Articles

MP39-06 ENHANCED CELL PROLIFERATION AND INCREASED RESISTANCE TO MAMMALIAN TARGET OF RAPAMYCIN INHIBITORS BY INHIBITION OF 4E-BINDING PROTEIN 1 EXPRESSION IN HUMAN RENAL CELL CARCINOMA ACHN CELLS

You have accessJournal of UrologyKidney Cancer: Basic Research I1 Apr 2015MP39-06 ENHANCED CELL PROLIFERATION AND INCREASED RESISTANCE TO MAMMALIAN TARGET OF RAPAMYCIN INHIBITORS BY INHIBITION OF 4E-BINDING PROTEIN 1 EXPRESSION IN HUMAN RENAL CELL CARCINOMA ACHN CELLS Akira Miyazaki, Hideaki Miyake, and Masato Fujisawa Akira MiyazakiAkira Miyazaki More articles by this author , Hideaki MiyakeHideaki Miyake More articles by this author , and Masato FujisawaMasato Fujisawa More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.756AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES 4E-binding protein 1 (4E-BP1), is an eukaryotic initiation factor 4E (eIF4E) binding protein, playing a critical role in the control of protein synthesis, cell growth and survival. When 4E-BP1 binds to eIF4E, this complex is demonstrated to suppress the cap-mediated translation, resulting in the induction of apoptosis in various types of cancer cells. The objective of this study was to clarify the functional significance of 4E-BP1 in the regulation of cell proliferation and sensitivity to proapoptotic stimuli in human renal cell carcinoma (RCC) ACHN cells. METHODS We initially established ACHN, in which the expression vector containing short hairpin RNA (shRNA) targeting 4E-BP1, was introduced (ACHN/sh-4E-BP1). Changes in the growth and sensitivity to several molecular targeted agents in ACHN/sh-4E-BP1 were compared with those in ACHN transfected with control vector alone (ACHN/C) both in vitro and in vivo. We also evaluated the expression of key molecules associated with apoptosis and signal transduction in ACHN sublines by Western blot analyses. RESULTS In vitro growth of ACHN/sh-4E-BP1 was significantly faster than that in ACHN/C. Expression levels of phosphorylated (p)-eIF4E, c-Myc, cyclin D1, p-p44-42 mitogen activated kinase (MAPK), p-mammalian target of rapamycin (mTOR), Bcl-2 and Mcl-1 in ACHN/sh-4E-BP1 were significantly upregulated compared with those in ACHN/C. Furthermore, ACHN/sh-4E-BP1 showed significantly higher sensitivities to temsirolimus and everolimus, but not those to sunitibi and axitiniv; that is, the IC50 of temsirolimus and everolimus in ACHN/sh-4E-BP1 were approximately 5- and 10-times, respectively, as high as those in ACHN/C. Sensitivities of both ACHN sublines to temsirolimus and everolimus became similar by additional treatment with a pecific inhibitor of c-Myc or MAPK. In vivo, the growth of ACHN/sh-4E-BP1 tumors in nude mice was significantly faster than that of ACHN/C tumors. Administration of either temsirolimus or everolimus induced the growth inhibition of both ACHN/C and ACHN/sh-4E-BP1 tumors; however, the growth inhibitory effects of these mTOR inhibitors on ACHN/C tumors were more marked than those on ACHN/sh-4E-BP1 tumors. CONCLUSIONS These findings suggest that 4E-BP1 plays an important role in the growth as well as resistance to mTOR inhibitors in RCC cells through the regulation of major molecules mediating the signal transduction and apoptosis. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e456 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Akira Miyazaki More articles by this author Hideaki Miyake More articles by this author Masato Fujisawa More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

172 RNA interference-mediated knock-down of CHD1 in human prostate xenograft tumours alters tumour growth and metastatic behaviour

growth of PC3/sh-4E-BP1 tumors in nude mice was significantly faster than that of PC3/C tumors. Administration of docetaxel induced the growth inhibition of both PC3/C and PC3/sh-4E-BP1 tumors; however, the growth inhibitory effect of docetaxel on PC3/C tumors was more marked than that on PC3/sh-4E-BP1 tumors. CONCLUSIONS: These findings suggest that 4E-BP1 plays an important role in the growth as well as resistance to proapoptotic therapeutic stimuli in CRPC cells through the regulation of major molecules mediating the signal transduction and apoptosis.

Thrombospondin-1 induces differential response in human corneal and conjunctival epithelial cells lines under in vitro inflammatory and apoptotic conditions

Recently, thrombospondin-1 (TSP-1) has been reported to be critical for maintaining a healthy ocular surface. The purpose of the study was to characterize the expression of TSP-1 and of its receptors CD36 and CD47 in corneal and conjunctival epithelial cells and determine the effect of exogenous TSP-1 treatment on these cells, following the induction of inflammation- and apoptosis-related changes. The expression of TSP-1, CD36 and CD47 by corneal and conjunctival cell lines was firstly characterized by ELISA, immunofluorescence analysis, Western blotting and reverse transcription polymerase chain reaction (RT-PCR). Benzalkonium chloride (BAC) exposure for 5 or 15 min was used as pro-inflammatory and pro-apoptotic stimulus for corneal or conjunctival epithelial cells, respectively. To analyze inflammation and apoptosis-related changes, IL-6 and TGF-β2 secretion determined by ELISA was used as inflammatory markers, while activated caspase-3/7 levels and cell viability, determined by CellEvent™ Caspase-3/7 Green Detection Reagent and XTT cytotoxicity assay, respectively, were used as apoptotic markers. Changes in CD36 and CD47 mRNA expression were quantified by real time RT-PCR. Corneal epithelial cells secreted and expressed higher protein levels of TSP-1 than conjunctival epithelial cells, although TSP-1 mRNA expression levels were similar and had lower CD36 and CD47, both at protein and mRNA levels. Both cell lines responded to exogenous TSP-1 treatment increasing CD36 at protein and mRNA levels. Blocking experiments revealed a predominance of TSP-1/CD47 rather than TSP-1/CD36 interactions to up-regulate CD36 levels in conjunctival epithelial cells, but not in corneal epithelial cells. BAC exposure increased IL-6 secretion and caspase-3/7 levels and decreased cell viability in both, corneal and conjunctival epithelial cells. Moreover, BAC exposure increased latent TGF-β2 levels in conjunctival epithelial cells. Interestingly, CD36 mRNA expression was down-regulated after BAC exposure in both cell lines. Exogenous TSP-1 treatment reduced TGF-β2 up-regulated levels by BAC exposure in conjunctival epithelial cells and less pronounced reduced IL-6 in BAC-exposed corneal epithelial cells. The effect on CD36 and CD47 regulation was less pronounced or even opposite depending on the inflammation- and apoptosis-related markers tested. Our results show evidence of the capacity of corneal and conjunctival epithelial cells to respond to TSP-1 via CD36 or CD47. Experimental simulation of inflammation- and apoptosis-related conditions changed the effects differentially elicited by TSP-1 on corneal and conjunctival epithelial cells, suggesting an unexpected and relevant contribution of TSP-1 on ocular surface homeostasis regulation.

Identification of cell populations in splenocytes of Nile Tilapia (Oreochromis niloticus) and functional parameters of lymphocytes through flow cytometry.

Identification of cell populations in splenocytes of Nile Tilapia (Oreochromis niloticus) and functional parameters of lymphocytes through flow cytometry.

Selumetinib produces a central core of apoptosis in breast cancer bone metastases in mice.

Bone is a common site for metastatic colonization in patients with breast cancer, hence the importance of identifying new treatments for this disease. Preclinical studies of bone metastases have commonly employed MDA-MB-231 cells that possess an activated KRAS allele. While activating RAS mutations are relatively rare in human breast cancer, increased RAS-RAF-MEK pathway activity is common in high-grade breast cancers. To study the consequences of MEK inhibition on bone metastases stemming from the intra-cardiac injection of luciferase-expressing MDA-MB-231 cells in mice, we used the MEK inhibitor AZD6244 (Selumetinib). We found that AZD6244 treatment caused decreased tumor bioluminescence that was associated with cavitation of the bone metastases, owing to apoptosis of cells specifically within the central region of the bone lesions. Hypothesizing that the latter effect was due to the increased sensitivity of poorly perfused regions to pro-apoptotic stimuli, we found that the combination of serum deprivation and AZD6244 led to dramatic induction pf MDA-MB-231 apoptosis in vitro. Our results suggest that MEK inhibition may be a strategy for triggering cell death within the hypoperfused, oxygen and nutrient poor regions of tumors with activated RAS alleles.

Novel cytoprotective inhibitors for apoptotic endonuclease G.

Apoptotic endonuclease G (EndoG) is responsible for DNA fragmentation both during and after cell death. Previous studies demonstrated that genetic inactivation of EndoG is cytoprotective against various pro-apoptotic stimuli; however, specific inhibitors for EndoG are not available. In this study, we have developed a high-throughput screening assay for EndoG and have used it to screen a chemical library. The screening resulted in the identification of two potent EndoG inhibitors, PNR-3-80 and PNR-3-82, which are thiobarbiturate analogs. As determined by their IC₅₀s, the inhibitors are more potent than ZnCl₂ or EDTA. They inhibit EndoG at one or two orders of magnitude greater than another apoptotic endonuclease, DNase I, and do not inhibit the other five tested cell death-related enzymes: DNase II, RNase A, proteinase, lactate dehydrogenase, and superoxide dismutase 1. Exposure of natural EndoG-expressing 22Rv1 or EndoG-overexpressing PC3 cells rendered them significantly resistant to Cisplatin and Docetaxel, respectively. These novel EndoG inhibitors have the potential to be utilized for amelioration of cell injuries in which participation of EndoG is essential.

Inhibitor of apoptosis proteins (IAPs) may be effective therapeutic targets for treating endometriosis.

What is the role of the inhibitor of apoptosis proteins (IAPs) in human endometriotic tissues and a mouse model of endometriosis? Four IAP proteins were expressed in endometriotic tissue indicating IAPs may be a key factor in the pathogenesis and progression of endometriosis. Overexpression of IAPs protects against a number of proapoptotic stimuli. IAPs (c-IAP1, c-IAP2, XIAP and Survivin) are expressed in human ectopic endometrial stromal cells (ESCs) from ovarian endometriomas. Forty-eight women with or without ovarian endometrioma are included in this study. BALB/c mice (n = 24) were used for the mouse endometriosis model. Mice with surgically induced endometriosis were treated with an IAP antagonist (BV6) for 4 weeks. Human ectopic endometrial tissues from chocolate cysts and eutopic endometrial tissue were collected. ESCs were enzymatically isolated from these tissues. ESC proliferation was examined by 5-bromo-2'-deoxyuridine-enzyme-linked immunosorbent assay. IAPs expression in tissue derived from eutopic endometria and chocolate cysts was evaluated using real-time RT-PCR and immunohistochemistry. A homologous mouse endometriosis model was established by transplanting donor mouse uterine tissue into the abdominal cavities of recipient mice. After treating the mice with BV6 (i.p. 10 mg/ml), the extent of endometriosis-like lesions in mice was measured and proliferative activity assessed by Ki67 staining. All experiments were repeated a minimum of three times. IAP (c-IAP1, c-IAP2, XIAP and Survivin) mRNA and protein in human ectopic endometrial tissues were expressed at higher levels than in eutopic endometrial tissues (P < 0.05). All four IAPs proteins were expressed in mouse endometriosis-like implants. BV6 inhibited BrdU incorporation of human ESCs (P < 0.05 versus control). BV6 also decreased the total number, weight, surface area and Ki67 positive cells in the endometriosis-like lesions in the mice (P < 0.05 versus control). Endometriotic lesions were surgically induced in mice by transplanting mouse uterine tissue only, not human pathological endometriotic tissue. Furthermore, the effects of BV6 on human ESCs and mouse endometriosis-like lesions may differ between the species. Our data support the hypothesis that IAPs are involved in the development of endometriosis, and therefore an inhibitor of IAPs has potential as a novel treatment for endometriosis. This work was supported by KAKENHI (Japan Society for the Promotion of Science, Grant-in-Aid: to F.T.; 21592098 and to T.H.; 24659731) and Yamaguchi Endocrine Research Foundation. The authors have no conflicts of interest to disclose.

Parkin Sensitizes toward Apoptosis Induced by Mitochondrial Depolarization through Promoting Degradation of Mcl-1

Mitochondrial depolarization promotes Parkin- and PTEN-induced kinase 1 (PINK1)-dependent polyubiquitination of multiple proteins on mitochondrial outer membranes, resulting in the removal of defective mitochondria via mitophagy. Because Parkin mutations occur in Parkinson's disease, a condition associated with the death of dopaminergic neurons in the midbrain, wild-type Parkin is thought to promote neuronal survival. However, here we show that wild-type Parkin greatly sensitized toward apoptosis induced by mitochondrial depolarization but not by proapoptotic stimuli that failed to activate Parkin. Parkin-dependent apoptosis required PINK1 and was efficiently blocked by prosurvival members of the Bcl-2 family or knockdown of Bax and Bak.Upon mitochondrial depolarization, the Bcl-2 family member Mcl-1 underwent rapid Parkin- and PINK1-dependent polyubiquitination and degradation, which sensitized toward apoptosis via opening of the Bax/Bak channel. These data suggest that similar to other sensors of cell stress, such as p53, Parkin has cytoprotective (mitophagy) or cytotoxic modes (apoptosis), depending on the degree of mitochondrial damage.

Caspase-9 mediates Puma activation in UCN-01-induced apoptosis.

The protein kinase inhibitor 7-hydroxystaurosporine (UCN-01) is one of the most potent and frequently used proapoptotic stimuli. The BH3-only molecule of Bcl-2 family proteins has been reported to contribute to UCN-01-induced apoptosis. Here we have found that UCN-01 triggers Puma-induced mitochondrial apoptosis pathway. Our data confirmed that Akt-FoxO3a pathway mediated Puma activation. Importantly, we elucidate the detailed mechanisms of Puma-induced apoptosis. Our data have also demonstrated that caspase-9 is a decisive molecule of Puma induction after UCN-01 treatment. Caspase-9 mediates apoptosis through two kinds of feedback loops. On the one hand, caspase-9 enhances Puma activation by cleaving Bcl-2 and Bcl-xL independent of caspase-3. On the other hand, caspase-9 directly activated caspase-3 in the presence of caspase-3. Caspase-3 could cleave XIAP in an another positive feedback loop to further sensitize cancer cells to UCN-01-induced apoptosis. Therefore, caspase-9 mediates Puma activation to determine the threshold for overcoming chemoresistance in cancer cells.

Enterococcus faecalis infection activates phosphatidylinositol 3-kinase signaling to block apoptotic cell death in macrophages.

Apoptosis is an intrinsic immune defense mechanism in the host response to microbial infection. Not surprisingly, many pathogens have evolved various strategies to manipulate this important pathway to benefit their own survival and dissemination in the host during infection. To our knowledge, no attempts have been made to explore the host cell survival signals modulated by the bacterium Enterococcus faecalis. Here, we show for the first time that during early stages of infection, internalized enterococci can prevent host cell (RAW264.7 cells, primary macrophages, and mouse embryonic fibroblasts [MEFs]) apoptosis induced by a wide spectrum of proapoptotic stimuli. Activation of caspase 3 and cleavage of the caspase 3 substrate poly(ADP-ribose) polymerase were inhibited in E. faecalis-infected cells, indicating that E. faecalis protects macrophages from apoptosis by inhibiting caspase 3 activation. This antiapoptotic activity in E. faecalis-infected cells was dependent on the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, which resulted in the increased expression of the antiapoptotic factor Bcl-2 and decreased expression of the proapoptotic factor Bax. Further analysis revealed that active E. faecalis physiology was important for inhibition of host cell apoptosis, and this feature seemed to be a strain-independent trait among E. faecalis isolates. Employing a mouse peritonitis model, we also determined that cells collected from the peritoneal lavage fluid of E. faecalis-infected mice showed reduced levels of apoptosis compared to cells from uninfected mice. These results show early modulation of apoptosis during infection and have important implications for enterococcal pathogenesis.

Ferrocifen derivatives that induce senescence in cancer cells: selected examples

Platinum coordination complexes represent an important class of anti-tumor agents. Due to recognized drawbacks, research into other types of metallodrugs has been diversified with the aim of finding new chemical entities with alternative mechanisms of action to overcome classical chemoresistance. P5 and DP1, two closely related ferrocenyl complexes bearing a similar ferrocenyl-ene-phenyl motif and displaying marked differences in their conformations and oxidation state versatility, were assayed in cancer cell models characterized by various sensitivities to pro-apoptotic stimuli. P5 and DP1 exert growth inhibitory effects between 0.5 and 10μM against glioma and melanoma cells including pluripotent stem-like cells. These effects are due, at least partly, to senescence induction with typical SA-β-galactosidase staining and senescence-associated secretory phenotype (SASP) as measured by the secretion of IL-1α, IL-1β, IL-6, IL-8 and TNF-α. Regulation of these cytokines' secretion may be related to AP-1 and other transcription factors unrelated to senescence. An in vivo graft of B16F10 cells after in vitro pre-incubation with DP1 or P5 led to increased survival in mice. In conclusion, P5 and DP1 ferrocenyl complexes induce senescence in various cancer cell models associated with distinct sensitivity to pro-apoptotic stimuli.

Regulation of mitochondrial apoptosis by Pin1 in cancer and neurodegeneration

Mitochondria are sensitive and efficient organelles that regulate essential biological processes including: energy metabolism, decoding and transduction of intracellular signals, and balance between cell death and survival. Of note, dysfunctions in mitochondrial physiology are a general hallmark of cancer cells, leading to transformation-related features such as altered cellular metabolism, survival under stress conditions and reduced apoptotic response to chemotherapy. Mitochondrial apoptosis is a finely regulated process that derives from activation of multiple signaling networks. A crucial biochemical requirement for transducing pro-apoptotic stimuli is represented by kinase-dependent phosphorylation cascades. In this context a pivotal role is played by the prolyl-isomerase Pin1, which translates Ser/Thr-Pro phosphorylation into conformational changes able to modify the activities of its substrates. In this review we will discuss the impact of Pin1 in regulating various aspects of apoptosis in different biological contexts with particular emphasis on cancer and neurodegenerative diseases.

Synthesis and anti-cancer activity of naturally occurring 2,5-diketopiperazines

Three naturally occurring oxyprenylated diketopiperazines were synthesized and preliminarily tested as growth inhibitory agents in vitro against various cancer cell lines. The compounds were tested on six human cancer cell lines with different sensitivity to proapoptotic stimuli using the MTT colorimetric assay. The data revealed that of the chemicals under study only deoxymicelianamide (11) displayed the highest activity, recording mean IC50 growth inhibitory values ranging from 2 to 23μM. A comparative study with the non-geranylated saturated derivative of (11) revealed the importance of the presence of the geranyloxy side chain and the exocyclic 2,5-DPK double bond moiety for the observed activity.

The small heat shock protein B8 (HSPB8) confers resistance to bortezomib by promoting autophagic removal of misfolded proteins in multiple myeloma cells.

Velcade is one of the inescapable drug to treat patient suffering from multiple myeloma (MM) and resistance to this drug represents a major drawback for patients. However, the mechanisms underlying velcade resistance remain incompletely understood. We derived several U266 MM cell clones that resist to velcade. U266-resistant cells were resistant to velcade-induced cell death but exhibited a similar sensitivity to various proapoptotic stimuli. Careful analysis of proteosomal subunits and proteasome enzymatic activities showed that neither the composition nor the activity of the proteasome was affected in velcade-resistant cells. Elimination of velcade-induced poly-ubiquitinated proteins and protein aggregates was drastically stimulated in the resistant cells and correlated with increased cell survival. Inhibition of the lysosomal activity in velcade-resistant cells resulted in an increase of cell aggregates and decrease survival, indicating that aggregates are eliminated through lysosomal degradation. In addition, pangenomic profiling of velcade-sensitive and resistant cells showed that the small heat shock protein HSPB8 was overexpressed in resistant cells. Finally, gain and loss of function experiment demonstrated that HSPB8 is a key factor for velcade resistance. In conclusion, HSPB8 plays an important role for the elimination of aggregates in velcade-resistant cells that contributes to their enhanced survival.

Detection of Caspase Activity Using Antibody-Based Techniques

A number of antibodies have been generated that recognize caspases from mammalian model organisms. These include antibodies that recognize specific caspase pro-forms and others that bind caspase cleavage fragments. These antibodies are excellent reagents for identifying which executioner caspases have been activated following application or induction of a specific apoptotic stimulus. This approach is more difficult to use with initiator caspases, however, because cleavage does not necessarily correlate with caspase activation. In this protocol, cultured cells are treated with a proapoptotic stimulus, and then protein lysates are prepared from the treated cells. The proteins are then separated by gel electrophoresis and transferred to a suitable membrane. The fragment-specific antibodies that recognize executioner caspases are used in a western analysis to determine the extent of activation and to aid in identifying which caspases have been activated.

Regulation of Endothelial Cell Barrier Function by Antibody-driven Affinity Modulation of Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1)

PECAM-1 is a 130-kDa member of the immunoglobulin (Ig) superfamily that is expressed on the surface of platelets and leukocytes, and at the intracellular junctions of confluent endothelial cell monolayers. Previous studies have shown that PECAM-1/PECAM-1 homophilic interactions play a key role in leukocyte transendothelial migration, in allowing PECAM-1 to serve as a mechanosensory complex in endothelial cells, in its ability to confer cytoprotection to proapoptotic stimuli, and in maintaining endothelial cell junctional integrity. To examine the adhesive properties of full-length PECAM-1 in a native lipid environment, we purified it from platelets and assembled it into phospholipid nanodiscs. PECAM-1-containing nanodiscs retained not only their ability to bind homophilically to PECAM-1-expressing cells, but exhibited regulatable adhesive interactions that could be modulated by ligands that bind membrane- proximal Ig Domain 6. This property was exploited to enhance the rate of barrier restoration in endothelial cell monolayers subjected to inflammatory challenge. The finding that the adhesive properties of PECAM-1 are regulatable suggests novel approaches for controlling endothelial cell migration and barrier function in a variety of vascular permeability disorders.

N-carbamidoyl-4-((3-ethyl-2,4,4-trimethylcyclohexyl)methyl)benzamide enhances staurosporine cytotoxic effects likely inhibiting the protective action of Magmas toward cell apoptosis.

We recently demonstrated that Magmas overexpression protects GH-secreting rat pitutitary adenoma cell lines from apoptosis by inhibiting cytochrome c release from mitochondria after treatment with staurosporine, strongly suggesting a role of Magmas in preventing apoptosis. The aim of this study was to produce a drug that, by inhibiting Tim16, may sensitize chemoresistant tumor cell to proapoptotic stimuli. We synthesized six compounds and challenged their sensitizing effects toward the proapoptotic effects of staurosporine in the TT cell line, derived from a human medullary thyroid carcinoma. We found that compound 5, devoid of the planarity in the aliphatic part of the lead 1, is not cytotoxic but enhances the proapoptotic effects of staurosporine by reducing MMP activation. Compound 5 may be useful for cancer treatment in association with chemotherapeutic drugs, possibly allowing a reduction of the chemotherapeutic agent effective dose.

Abstract 342: The Antiapoptotic Function of APEX Nuclease (Multifunctional DNA Repair Enzyme) 1 in Endothelial Cells Is Linked to Changes in Transcription Factor Activities and Independent of Its DNA Repair Domain

APEX nuclease (multifunctional DNA repair enzyme) 1 (APEX1) has both DNA repair and redox regulatory activity. Interestingly, APEX1 is also able to modulate transcription factor reduction, a prerequisite for DNA binding, independent of its intrinsic redox activity, probably by recruiting other reducing molecules like Thioredoxin-1 (TXN). APEX1 has been shown to exert anti-apoptotic properties in different cell lines and is localized in the cytosol and in the nucleus. So far it has not been studied whether APEX1 acts anti-apoptotic in primary human endothelial cells (EC) and whether this is dependent on its nuclear or cytosolic localization. After cloning human APEX1 and overexpression in EC we demonstrated that it inhibits not only basal but also H2O2-induced apoptosis. To further characterize the anti-apoptotic properties of APEX1 we generated mutants that lacked the C-terminal DNA repair domain (APEX1ΔC 1-127) or the N-terminal nuclear localization signal (NLS) (APEX1ΔN 21-318). We first analyzed the localization of the mutants by immunostaining. APEX1wt and APEX1ΔC 1-127 showed a predominant nuclear staining whereas APEX1ΔN 21-318 was mainly localized in the cytosol. With respect to the anti-apoptotic properties, only the mutant with intact NLS and redox domain APEX1ΔC 1-127 prevented apoptosis both under basal conditions as well as after H2O2 treatment similar to APEX1wt. The association between nuclear localization of APEX1wt and its anti-apoptotic properties in EC suggests that this function is linked to changes in transcription factor activities. It is known that the p65 subunit of the transcription factor complex NF-kB is activated by pro-apoptotic stimuli in EC. Using a specific DNA binding assay we could show that overexpression of APEX1wt and APEX1ΔC 1-127 reduced DNA binding of p65, whereas the NLS-deficient mutant had no effect. In conclusion, the anti-apoptotic activity of APEX1 in EC critically depends on its nuclear localization and redox-activity, is independent of its DNA repair domain and is at least partially due to suppression of NF-kB dependent, pro-apoptotic gene expression programs. In future studies we will analyze the impact of APEX1 on other transcription factors in concert with TXN.

Extracellular ADP prevents neuronal apoptosis via activation of cell antioxidant enzymes and protection of mitochondrial ANT-1

Apoptosis in neuronal tissue is an efficient mechanism which contributes to both normal cell development and pathological cell death. The present study explores the effects of extracellular ADP on low [K+]-induced apoptosis in rat cerebellar granule cells. ADP, released into the extracellular space in brain by multiple mechanisms, can interact with its receptor or be converted, through the actions of ectoenzymes, to adenosine. The findings reported in this paper demonstrate that ADP inhibits the proapoptotic stimulus supposedly via: i) inhibition of ROS production during early stages of apoptosis, an effect mediated by its interaction with cell receptor/s. This conclusion is validated by the increase in SOD and catalase activities as well as by the GSSG/GSH ratio value decrease, in conjunction with the drop of ROS level and the prevention of the ADP protective effect by pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), a novel functionally selective antagonist of purine receptor; ii) safeguard of the functionality of the mitochondrial adenine nucleotide-1 translocator (ANT-1), which is early impaired during apoptosis. This effect is mediated by its plausible internalization into cell occurring as such or after its hydrolysis, by means of plasma membrane nucleotide metabolizing enzymes, and resynthesis into the cell. Moreover, the findings that ADP also protects ANT-1 from the toxic action of the two Alzheimer's disease peptides, i.e. Aβ1–42 and NH2htau, which are known to be produced in apoptotic cerebellar neurons, further corroborate the molecular mechanism of neuroprotection by ADP, herein proposed.

ATP signaling complex is altered in muscular dystrophy and was partly recovered after nifedipine treatment (762.3)

Duchenne muscular dystrophy (DMD) is a common genetic disorder characterized by a severe muscle wasting caused by the absence of dystrophin. In mdx muscle fibers, we have shown that basal ATP release is increased and that high extracellular ATP is a pro‐apoptotic stimuli. We also have shown that Dihydropyridine receptors (DHPR) are needed for ATP release through pannexin‐1(Pnx1) channels. The aim of this work was to study the potential therapeutic effect of DHPR blockage by nifedipine in DMD. We used muscle fibers isolated from six‐week‐old normal and mdx mice that were treated with daily intraperitoneal injections of 1mg/Kg nifedipine for 1‐week. We studied differences in the interaction between DHPR and Pnx1by proximity ligation assay. In mdx fibers there was an increase in the number and volume of positive particles but not in the intensity of the positive reaction when compared to normal fibers. After the treatment with nifedipine, both number and volume of the positive reaction particles was normalized. This result correlates well with a decrease in both ATP release and intracellular resting Ca2+ concentration in mdx fibers after the treatment. Moreover, ATP signaling was no longer a pro‐apoptotic stimuli. Finally, nifedipine treatment increased muscle strength assessed by both the inverted grip‐hanging and forced swimming test. These data suggest that dihydropyridines can be used as a therapeutical tool to reduce muscle damage observed in dystrophic muscles.Grant Funding Source: Fondecyt 1110467, ACT1111, AFM14562, AT‐24110211