Anti-apoptotic Processes Research Articles

The Ameliorative Effects of L-2-Oxothiazolidine-4-Carboxylate on Acetaminophen-Induced Hepatotoxicity in Mice

The aim of the study was to investigate the ameliorative effects and the mechanism of action of l-2-oxothiazolidine-4-carboxylate (OTC) on acetaminophen (APAP)-induced hepatotoxicity in mice. Mice were randomly divided into six groups: normal control group, APAP only treated group, APAP + 25 mg/kg OTC, APAP + 50 mg/kg OTC, APAP + 100 mg/kg OTC, and APAP + 100 mg/kg N-acetylcysteine (NAC) as a reference control group. OTC treatment significantly reduced serum alanine aminotransferase and aspartate aminotransferase levels in a dose dependent manner. OTC treatment was markedly increased glutathione (GSH) production and glutathione peroxidase (GSH-px) activity in a dose dependent manner. The contents of malondialdehyde and 4-hydroxynonenal in liver tissues were significantly decreased by administration of OTC and the inhibitory effect of OTC was similar to that of NAC. Moreover, OTC treatment on APAP-induced hepatotoxicity significantly reduced the formation of nitrotyrosin and terminal deoxynucleotidyl transferase dUTP nick end labeling positive areas of liver tissues in a dose dependent manner. Furthermore, the activity of caspase-3 in liver tissues was reduced by administration of OTC in a dose dependent manner. The ameliorative effects of OTC on APAP-induced liver damage in mice was similar to that of NAC. These results suggest that OTC has ameliorative effects on APAP-induced hepatotoxicity in mice through anti-oxidative stress and anti-apoptotic processes.

Hyperlipidemia Modifies Innate Immune Responses to Lipopolysaccharide via the TLR-NF-κB Signaling Pathway

The objective of this study was to investigate the effect of hyperlipidemia (HLP) on innate immune responses to lipopolysaccharide (LPS). Male New Zealand white rabbits were fed a normal diet (ND) or a high-fat diet (HFD) for 8 weeks. In vivo, the rabbits were injected intravenously with LPS for 24 h. In vitro, peripheral mononuclear cells were collected and stimulated (or unstimulated) with LPS for 24 h. Assay results were analyzed with one-way ANOVA or an equivalent non-parametric test. A P value of 0.05 was considered statistically significant. Despite having no influence in body weight, the HFD intake significantly increased serum lipids, C-reactive protein (CRP), nuclear factor (NF)-κB subunit p65, Toll-like receptor (TLR)-4, SR-A and FAS. Although we found increased circulating tumor necrosis factor (TNF)-α, interleukin (IL)-6, CRP, IL-1β, and IL-10 in the ND-fed rabbits, no significant difference was found in the LPS-stimulated production of TNF-α, IL-6, and CRP in the HFD-fed rabbits. The macrophages harvested from the HFD-fed rabbits developed a blunted inflammatory response, with lower mRNA expression of TNF-α, IL-6, CRP, TLR-4, SR-A, FAS, and Bcl-2 than that expressed by the ND group. In the HFD-fed animals, LPS incubation decreased NF-κB subunit p65 expression, whereas the cytoplasmic phosphorylation of the inhibitor of NF-κB protein was enhanced. These data indicate that HLP displayed a form of innate immune paralysis, including reduced pro- and anti-inflammatory cytokine release to external stimulus, which was related to the altered TLR-NF-κB signaling pathway and altered pro- and anti-apoptotic processes in macrophages.

Hypoxia and Integrin-Mediated Epithelial Restitution during Mucosal Inflammation

Epithelial damage and loss of intestinal barrier function are hallmark pathologies of the mucosal inflammation associated with conditions such as inflammatory bowel disease. In order to resolve inflammation and restore intestinal integrity the mucosa must rapidly and effectively repair the epithelial barrier. Epithelial wound healing is a highly complex and co-ordinated process and the factors involved in initiating intestinal epithelial healing are poorly defined. In order for restitution to be successful there must be a balance between epithelial cell migration, proliferation, and differentiation within and adjacent to the inflamed area. Endogenous, compensatory epithelial signaling pathways are activated by the changes in oxygen tensions that accompany inflammation. These signaling pathways induce the activation of key transcription factors, governing anti-apoptotic, and proliferative processes resulting in epithelial cell survival, proliferation, and differentiation at the site of mucosal inflammation. In this review, we will discuss the primary processes involved in epithelial restitution with a focus on the role of hypoxia-inducible factor and epithelial integrins as mediators of epithelial repair following inflammatory injury at the mucosal surface.

The influence of the pleiotropic action of erythropoietin and its derivatives on nephroprotection.

Erythropoietin (EPO) is traditionally described as a hematopoietic cytokine or growth hormone regulating proliferation, differentiation, and survival of erythroid progenitors. The use of EPO in patients with chronic kidney disease (CKD) was a milestone achievement in the treatment of anemia. However, EPO involves some degree of risk, which increases with increasing hemoglobin levels. A growing number of studies have assessed the renoprotective effects of EPO in acute kidney injury (AKI) or CKD. Analysis of the biological effects of erythropoietin and pathophysiology of CKD in these studies suggests that treatment with erythropoiesis-stimulating agents (ESAs) may exert renoprotection by pleiotropic actions on several targets and directly or indirectly slow the progression of CKD. By reducing ischemia and oxidative stress or strengthening anti-apoptotic processes, EPO may prevent the development of interstitial fibrosis and the destruction of tubular cells. Furthermore, it could have a direct protective impact on the integrity of the interstitial capillary network through its effects on endothelial cells and promotion of vascular repair, or modulate inflammation response. Thus, it is biologically plausible to suggest that correcting anemia with ESAs could slow the progression of CKD.The aim of this article is to discuss these possible renoprotection mechanisms and provide a comprehensive overview of erythropoietin and its derivatives.

Early passage neonatal and adult keratinocytes are sensitive to apoptosis induced by infection with an ICP27-null mutant of herpes simplex virus 1

Herpes simplex virus 1 (HSV-1) is a enveloped, double stranded DNA virus that is the causative agent of various diseases including cold sores, encephalitis, and ocular keratitis. Previous research has determined that HSV-1 modulates cellular apoptotic pathways. Apoptosis is triggered in infected cells early in infection; however, later in the infection the apoptotic response is suppressed due to the expression of several viral apoptotic antagonists. This sets us a delicate balance between pro- and anti-apoptotic processes during the lytic phase of infection. Several studies have demonstrated that the apoptotic balance can be shifted during infection of certain cell types, leading to apoptosis of the infected cells (HSV-1-dependent apoptosis). For example, HEp-2 cells infected with an ICP27-null recombinant HSV-1 virus undergo HSV-1-dependent apoptosis. Differences in the sensitivity to HSV-1-dependent apoptosis have been revealed. Although many tumor cells have been found to be highly sensitive to this apoptotic response, with the exception hematological cells, all primary human cells tested prior to this study have been shown to be resistant to HSV-1-dependent apoptosis. Here, we demonstrate that early passage neonatal and adult human keratinocytes, which are usually the first cells to encounter HSV-1 in human infection and support the lytic stage of the life cycle, display membrane blebbing and ballooning, chromatin condensation, caspase activation, and cleavage of cellular caspase substrates when infected with an ICP27-null recombinant of HSV-1. Furthermore, caspase activation is needed for the efficient apoptotic response. These results suggest that apoptotic machinery may be a target for modulating HSV-disease in patients.

Constitutive Notch2 signaling in neural stem cells promotes tumorigenic features and astroglial lineage entry

Recent studies identified a highly tumorigenic subpopulation of glioma stem cells (GSCs) within malignant gliomas. GSCs are proposed to originate from transformed neural stem cells (NSCs). Several pathways active in NSCs, including the Notch pathway, were shown to promote proliferation and tumorigenesis in GSCs. Notch2 is highly expressed in glioblastoma multiforme (GBM), a highly malignant astrocytoma. It is therefore conceivable that increased Notch2 signaling in NSCs contributes to the formation of GBM. Here, we demonstrate that mice constitutively expressing the activated intracellular domain of Notch2 in NSCs display a hyperplasia of the neurogenic niche and reduced neuronal lineage entry. Neurospheres derived from these mice show increased proliferation, survival and resistance to apoptosis. Moreover, they preferentially differentiate into astrocytes, which are the characteristic cellular population of astrocytoma. Likewise, we show that Notch2 signaling increases proliferation and resistance to apoptosis in human GBM cell lines. Gene expression profiling of GBM patient tumor samples reveals a positive correlation of Notch2 transcripts with gene transcripts controlling anti-apoptotic processes, stemness and astrocyte fate, and a negative correlation with gene transcripts controlling proapoptotic processes and oligodendrocyte fate. Our data show that Notch2 signaling in NSCs produces features of GSCs and induces astrocytic lineage entry, consistent with a possible role in astrocytoma formation.

Dynamics of signaling, cytoskeleton and cell cycle regulation proteins in glioblastoma cells after sub-lethal photodynamic treatment: Antibody microarray study

BackgroundPhotodynamic therapy (PDT) that induces oxidative stress and cell death is used for tumor destruction in oncology. To characterize early molecular events in photosensitized glioblastoma cells, we studied expression of 224 proteins after sublethal PDT that doesn't kill but wounds cells. MethodsCultured glioblastoma D54Mg cells were photosensitized with 5-aminolevulinic acid so that cell survival was 95–100%. At following 0.5–5.5h protein expression and phosphorylation was assayed using proteomic antibody microarrays. ResultsWithin the first post-treatment hour we observed phosphorylation of protein kinase Raf, adhesion-related kinases FAK and Pyk2, and microtubule-associated protein tau. Protein kinase Cγ and microtubule-associated protein MAP-1B were overexpressed. Dystrophin, calponin, and vinculin, components of the actin cytoskeleton scaffold, microtubule-associated proteins MAP2 and CNP, cytokeratins 4 and 7 were down-regulated that indicated changes in adhesion and cell shape. Down-regulation of cyclins A, D1 and D3, c-Myc, checkpoint proteins chk1/2 and up-regulation of Smad4 could arrest the cell cycle. Overexpression of Bcl-xL and down-regulation of caspase 9 demonstrated anti-apoptotic response. At 2h post-treatment protein expression changed lesser but at 5.5h levels of PKCγ and β-synuclein and phosphorylation of Raf, FAK, Pyk2, and tau increased again. ConclusionsSub-lethal PDT induces complex response of glioblastoma cells including changes in activity and expression of proteins involved in adhesion-mediated signaling, signal transduction, cytoskeleton remodeling, cell cycle regulation and anti-apoptotic processes. General significanceMultiple reactions of various cellular subsystems including adhesion, cytoskeleton, signal transduction, cell cycle, and apoptosis are integrated into the general cell response to a sublethal impact.

Вплив стрептозотоциніндукованого діабету та неповної глобальної ішемії мозку на апоптоз у тимусі щурів

The influence of incomplete global ischemia-reperfusion of the brain on the activity of pro- and antiapoptotic processes in the thymocytes by the intensity of p53 and Bcl-2 - proteins expressions has been investigated. It has been shown that in the cortical zone of thymus of rats with a four-month diabetes, as well as in the control rats and rats with diabetes mellitus after brain ischemia, the antiapoptotic processes are dominated. In the medullar zone of the gland in rats with diabetes, complicated and uncomplicated with brain ischemia-reperfusion the antiapoptotic mechanisms predominate too, but to a lesser extent than in the cortical zone.

Untangling the Roles of Anti-Apoptosis in Regulating Programmed Cell Death using Humanized Yeast Cells

Genetically programmed cell death (PCD) mechanisms, including apoptosis, are important for the survival of metazoans since it allows, among things, the removal of damaged cells that interfere with normal function. Cell death due to PCD is observed in normal processes such as aging and in a number of pathophysiologies including hypoxia (common causes of heart attacks and strokes) and subsequent tissue reperfusion. Conversely, the loss of normal apoptotic responses is associated with the development of tumors. So far, limited success in preventing unwanted PCD has been reported with current therapeutic approaches despite the fact that inhibitors of key apoptotic inducers such as caspases have been developed. Alternative approaches have focused on mimicking anti-apoptotic processes observed in cells displaying increased resistance to apoptotic stimuli. Hormesis and pre-conditioning are commonly observed cellular strategies where sub-lethal levels of pro-apoptotic stimuli lead to increased resistance to higher or lethal levels of stress. Increased expression of anti-apoptotic sequences is a common mechanism mediating these protective effects. The relevance of the latter observation is exemplified by the observation that transgenic mice overexpressing anti-apoptotic genes show significant reductions in tissue damage following ischemia. Thus strategies aimed at increasing the levels of anti-apoptotic proteins, using gene therapy or cell penetrating recombinant proteins are being evaluated as novel therapeutics to decrease cell death following acute periods of cell death inducing stress. In spite of its functional and therapeutic importance, more is known regarding the processes involved in apoptosis than anti-apoptosis. The genetically tractable yeast Saccharomyces cerevisiae has emerged as an exceptional model to study multiple aspects of PCD including the mitochondrial mediated apoptosis observed in metazoans. To increase our knowledge of the process of anti-apoptosis, we screened a human heart cDNA expression library in yeast cells undergoing PCD due to the conditional expression of a mammalian pro-apoptotic Bax cDNA. Analysis of the multiple Bax suppressors identified revealed several previously known as well as a large number of clones representing potential novel anti-apoptotic sequences. The focus of this review is to report on recent achievements in the use of humanized yeast in genetic screens to identify novel stress-induced PCD suppressors, supporting the use of yeast as a unicellular model organism to elucidate anti-apoptotic and cell survival mechanisms.

The Receptor That Tames the Innate Immune Response

Tissue injury, hypoxia and significant metabolic stress activate innate immune responses driven by tumor necrosis factor (TNF)-α and other proinflammatory cytokines that typically increase damage surrounding a lesion. In a compensatory protective response, erythropoietin (EPO) is synthesized in surrounding tissues, which subsequently triggers antiinflammatory and antiapoptotic processes that delimit injury and promote repair. What we refer to as the sequelae of injury or disease are often the consequences of this intentionally discoordinated, primitive system that uses a "scorched earth" strategy to rid the invader at the expense of a serious lesion. The EPO-mediated tissue-protective system depends on receptor expression that is upregulated by inflammation and hypoxia in a distinctive temporal and spatial pattern. The tissue-protective receptor (TPR) is generally not expressed by normal tissues but becomes functional immediately after injury. In contrast to robust and early receptor expression within the immediate injury site, EPO production is delayed, transient and relatively weak. The functional EPO receptor that attenuates tissue injury is distinct from the hematopoietic receptor responsible for erythropoiesis. On the basis of current evidence, the TPR is composed of the β common receptor subunit (CD131) in combination with the same EPO receptor subunit that is involved in erythropoiesis. Additional receptors, including that for the vascular endothelial growth factor, also appear to be a component of the TPR in some tissues, for example, the endothelium. The discoordination of the EPO response system and its relative weakness provide a window of opportunity to intervene with the exogenous ligand. Recently, molecules were designed that preferentially activate only the TPR and thus avoid the potential adverse consequences of activating the hematopoietic receptor. On administration, these agents successfully substitute for a relative deficiency of EPO production in damaged tissues in multiple animal models of disease and may pave the way to effective treatment of a wide variety of insults that cause tissue injury, leading to profoundly expanded lesions and attendant, irreversible sequelae.

Photodynamic Treatment Induces an Apoptotic Pathway Involving Calcium, Nitric Oxide, p53, p21-Activated Kinase 2, and c-Jun N-Terminal Kinase and Inactivates Survival Signal in Human Umbilical Vein Endothelial Cells

Photodynamic treatment (PDT) elicits a diverse range of cellular responses, including apoptosis. Previously, we showed that PDT stimulates caspase-3 activity, and subsequent cleavage and activation of p21-activated kinase 2 (PAK2) in human epidermal carcinoma A431 cells. In the current study, pretreatment with nitric oxide (NO) scavengers inhibited PDT-induced mitochondrial membrane potential (MMP) changes, activation of caspase-9, caspase-3, p21-activated protein kinase 2 (PAK2) and c-Jun N-terminal kinase (JNK), and gene expression of p53 and p21 involved in apoptotic signaling. Moreover, PAK2 activity was required for PDT-induced JNK activation and apoptosis. Inhibition of p53 mRNA expression using small interfering RNA (siRNA) additionally blocked activation of PAK2 and apoptosis induced by PDT. Importantly, our data also show that PDT triggers cell death via inactivation of ERK-mediated anti-apoptotic pathway. PDT triggers cell death via inactivation of the HSP90/multi-chaperone complex and subsequent degradation of Ras, further inhibiting anti-apoptotic processes, such as the Ras→ERK signal transduction pathway. Furthermore, we did not observe two-stage JNK activation for regulation of PAK2 activity in the PDT-induced apoptotic pathway in HUVECs, which was reported earlier in A431 cells. Based on the collective results, we have proposed a model for the PDT-triggered inactivation of the survival signal and apoptotic signaling cascade with Rose Bengal (RB), which sequentially involves singlet oxygen, Ca2+, NO, p53, caspase-9, caspase-3, PAK2, and JNK.

Potential targets for molecular imaging of apoptosis resistance in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers, which is mainly a concern in Southeast Asia. Apoptosis resistance in HCC is one of the significant factors for hepatocarcinogenesis and tumour progression. Recent advances of apoptosis resistance mechanisms in HCC could serve as potential targets for molecular imaging, which would be of considerable value to explore the molecular processes involved in HCC progression and to evaluate responses of certain anti-HCC therapies. Disruptions in the balance of anti-apoptotic and pro-apoptotic processes have been found to be involved in apoptosis resistance in HCC. Loss of response to death receptors, transformation of growth factor-β induced apoptosis, upregulation of anti-apoptotic Bcl-2 subgroup, as well as downregulation of pro-apoptotic Bax subgroup and BH3-only subgroup, are associated with apoptosis resistance in HCC. Mutation of p53 gene, dysregulation of NF-κB and survivin are also of interest because of their contribution to HCC development. In this review, the aim is to identify potential targets for molecular imaging of apoptosis resistance in HCC.

Advance in the Study of the Mechanisms Regulated by Sphingosine-1-Phosphate

Sphingosine-1-phosphate (S1P) is a bioactive lipid messenger in the cells that regulate gene expression and NF-KB signal pathway through unknown mechanisms. Recently, Cheng Luo, associate professor of DDDC in Shanghai Institute of Materia Medica, whose project was funded by the National Natural Science Foundation of China, joined in a research team led by Professor Sarah Spiegel of Virginia Commonwealth University. The team continuously made significant breakthroughs in understanding the regulation mechanism of Sphingosine-1-Phosphate. In September 2009, in a paper published on SCIENCE magazine (Science 2009, 325: 1254-7), they firstly demonstrated that S1P is a physiologically important regulator of histone deacetylases (HDACs), HDACs are direct intracellular targets of S1P. Furthermore, they identified the mechanism that S1P regulates gene expression through regulating the activity of HDACs. In June 24th, 2010, in another paper to be published on NATURE magazine (Nature 2010, June 24th, advance online publication) which reports the regulation of NF-KB signaling pathway by S1P. They demonstrate that S1P is the missing cofactor for TRAF2 (tumour-necrosis factor receptor-associated factor 2) and indicate a new paradigm for the regulation of lysine-63-linked poly-ubiquitination. The study also highlight the key role of SphK1 and its product S1P in TNF-α signalling and the canonical NF-KB activation pathway, and then play crucial role in inflammatory, antiapoptotic and immune processes. The identification of new mechanisms by which S1P regulates gene expression and TNF and NF-KB signaling pathway will light up the road to develop novel inhibitors that might be useful for treatment of cancer and inflammatory diseases.

Quantitative shotgun proteomic analysis of seminal plasma from men submitted to intracytoplasmic sperm injection (ICSI)

OBJECTIVE: The seminal plasma contains a number of proteins which participate in pathways leading to fertilization or which reflect testicular and post-testicular processes responsible for correct sperm production and maturation. Thus, we wished to verify if differential protein expression in the seminal plasma may predict outcome in ICSI cycles. DESIGN: Prospective case-control study. MATERIALS AND METHODS: Semen was obtained from 6 men being treated for infertility using ICSI – 3 who achieved pregnancy (group 1) in that cycle and 3 who did not (group 2). Seminal plasma was collected from an unused aliquot following centrifugation for removal of cellular components and frozen until analysis. Samples were pooled by group and submitted to nanoUPLC tandem nanoESI-MSE quantitative shotgun proteomics. Statistically identified proteins were analyzed for interactome networks using Cytoscape and classified according to Gene Ontology. RESULTS: A total of 222 proteins were identified, 96 differentially expressed between the three groups – 32 exclusively in group 1, 39 exclusively expressed in group 2, 14 overexpressed in group 1, and 11 overexpressed in group 2. Cytoscape analysis was performed using 1 degree of interaction, in order to verify both seminal plasma proteins and their putative interactions with sperm proteins. 30 clusters were observed. Gene Ontology analysis of these clusters demonstrated common biological processes to both groups, such as regulation of nitric oxide synthase activity, and positive regulation of signal transduction. Group 1 presented anti-apoptotic processes, while group 2 presented acute-phase response and pro-apoptotic processes. CONCLUSION: The use of a quantitative shotgun proteomics approach has increased sensitivity and the dynamic range for protein identification. The information from this study may allow us to (i) better understand the pathways leading to normal fertilization and allowing pregnancy, and (b) determine biomarkers for therapeutic intervention or sperm selection.

Sphingosine-1-phosphate is a missing cofactor for the E3 ubiquitin ligase TRAF2

TNF receptor-associated factor 2 (TRAF2) is a key component in NF-κB signaling triggered by TNF–α 1,2. Genetic evidence indicates that TRAF2 is necessary for polyubiquitination of receptor interacting protein 1 (RIP1) 3 that then serves as a platform for recruitment and stimulation of IκB kinase (IKK) leading to activation of the transcription factor NF-κB. Although TRAF2 is a RING domain ubiquitin ligase, direct evidence that TRAF2 catalyzes the ubiquitination of RIP1 is lacking. TRAF2 binds to sphingosine kinase 1 (SphK1) 4, one of the isoenzymes that generates the pro-survival lipid mediator sphingosine-1-phosphate (S1P) inside cells. Here we show that SphK1 and production of S1P is necessary for Lys 63-linked polyubiquitination of RIP1, phosphorylation of IKK and IκBα, and IκBα degradation, leading to NF-κB activation. Surprisingly, these responses were mediated by intracellular S1P independently of its cell surface G protein-coupled receptors. S1P specifically binds to TRAF2 at the N-terminal RING domain and stimulates its E3 ligase activity. S1P, but not dihydro-S1P, dramatically increased recombinant TRAF2-catalyzed Lys 63- but not Lys 48-linked polyubiquitination of RIP1 in vitro in the presence of the ubiquitin conjugating enzymes (E2) UbcH13 or UbcH5a. Our data reveal that TRAF2 is a novel intracellular target of S1P, and that S1P is the missing co-factor for TRAF2 E3 ubiquitin ligase activity, suggesting a new paradigm for regulation of Lys 63-linked polyubiquitination. These results also highlight the key role of SphK1 and its product S1P in TNF-α signaling and the canonical NF-κB activation pathway important in inflammatory, anti-apoptotic, and immune processes.

The effect of prenatal nicotine exposure on PDGFR-mediated anti-apoptotic mechanism in the caudal brainstem of developing rat

Maternal cigarette smoking is a major risk factor for sudden infant death syndrome (SIDS); however, the mechanism underlying this association is currently unknown. Prenatal nicotine exposure is accompanied by a decrease in the magnitude of hypoxic ventilatory depression, the component of hypoxic ventilatory response that activates the PDGF-β receptor (PDGFR) and its downstream anti-apoptotic cascade in the caudal brainstem (CB) of developing rats. In this study, we evaluated the effect of prenatal nicotine exposure on PDGFR activation and the subsequent activation of downstream anti-apoptotic processes through the Akt/BAD pathway. The 5-day timed-pregnant Sprague–Dawley rats underwent surgical implantation of an osmotic pump containing either normal saline (control) or a solution of nicotine tartrate. The CB was harvested from 5-day-old rat pups ( n = 8–10 for each time point) in each group after exposure to normoxia or hypoxic challenges with 10% O 2 for 5, 15, 30, 60 or 120 min. Immunoprecipitation and immunoblots of CB lysates revealed phosphorylation of PDGFR, Akt and BAD-136 during hypoxia in control pups. Prenatal nicotine exposure was associated with attenuation of these responses at all time points. Analysis of an early apoptotic marker in the CB revealed that activation of cleaved caspase-3 occurred only at 120 min of hypoxic exposure in the control. Prenatal nicotine exposure accelerated this response, causing early activation at 30 and 60 min. We conclude that prenatal nicotine exposure attenuates the phosphorylation of PDGFR, Akt and Bad-136 during hypoxia in the CB of developing rats. This modulation of anti-apoptotic cascades accelerates activation of the early apoptotic marker. We speculate that prenatal nicotine exposure affects apoptosis in the CB of developing animals and may increase the vulnerability of neural cells in the respiratory control area, a process that may underlie the association between maternal smoking and SIDS.

Abstract 5043: Effects of a novel PI3 kinase/mTOR inhibitor on proliferation and pAKT signaling in canine lymphoma

Abstract PF-04691503 is a dual inhibitor of both PI3K and mTOR, inhibits PI3K signaling in cancer cell lines, and exhibits in vitro and in vivo anti-proliferative activity in PI3K-pathway driven cell lines. It is a nanomolar inhibitor of all 4 isoforms of the catalytic subunit of PI3K and of both TORC1 and TORC2. Anti-cancer activity of the inhibitor is hypothesized to be through inhibition of survival, proliferative, and anti-apoptotic processes. Phosphorylation of the protein kinase, Akt, is associated with activation of the phosphatidyl 3-kinase (PI-3K)/mammalian target of rapamycin (mTOR) signaling pathway, which plays a role in cell proliferation (Witzig and Kaufmann 2006). Recent studies by Gulmann et al (2005) showed increased pAKT in human lymphoma samples, and those of Rassidakis et al (2005) suggested that inhibition of Akt phosphorylation (pAKT) may be of value in the treatment of lymphoma. Using flow cytometry we report that cells obtained from 10 of 11 lymph node biopsies of dogs with lymphoma exhibit detectable pAKT using a phospho-Akt (Ser473) antibody, compared to an IgG isotype control or a competative phospho-Akt (Ser473) blocking peptide. Cells from normal canine lymph nodes do not exhibit detectable pAKT. The majority of the pAKT signal was generated from lymphoblasts present in the malignant, but not in the normal, lymph nodes. In separate studies, lymph node cells obtained from healthy dogs and dogs with lymphoma were stimulated in vitro with the mitogen, Con A. The novel PI3K/mTOR dual inhibitor, PF-04691503, produced dose dependent inhibition of proliferation as exemplified in a dog with T-cell lymphoma (EC50 = 18 nM)) and in a normal dog (EC50 = 53 nM)). No pAKT signal could be detected in peripheral blood mononuclear cells, from normal or lymphoma patients stimulated with hu-IGF-1 (the endogenous ligand for PI3), Con-A or LPS. These data suggest that PI-3 kinase and pAKT (1) are activated in canine lymphoma, (2) play a role in the lymphoproliferation associated with this disease, (3) represent legitimate targets for therapeutic intervention in lymphoma, and (4) can be studied ‘translationally’ in dogs as a model for humans. Gulmann C, Espina V, Petricoin E, et al. Proteomic analysis of apoptotic pathways reveals prognostic factors in follicular lymphoma. Clin Cancer Res 11:5847-5855, 2005. Rassidakis GZ, Feretzaki M, Atwell C, et al. Inhibition of Akt increases p27 Kip1 levels and induces cell cycle arrest in anaplastic large cell lymphoma. Blood 105:827-829, 2005. Witzig TE and Kaufmann SH. Inhibition of phosphatidyl 3-kinase (PI-3K)/mammalian target of rapamycin (mTOR) pathway in hematologic malignancies. Current Treatment Options in Oncology 7:285-294, 2006. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5043.

Abstract 3492: A novel PI3K/mTOR dual inhibitor provides correlations of pharmacokinetic pharmacodynamic and tumor growth inhibition in a prostate PC3 xenograft model for application in clinical trials

Abstract PF-04691502 is a dual inhibitor of both PI3K and mTOR, inhibits PI3K signaling in cancer cell lines, and exhibits in vitro and in vivo anti-proliferative activity in PI3K-pathway driven cell lines. It is a nanomolar inhibitor of all 4 isoforms of the catalytic subunit of PI3K and of both TORC1 and TORC2. Anti-cancer activity of the inhibitor is hypothesized to be through inhibition of survival, proliferative, and anti-apoptotic processes. Its predicted human bioavalibility is roughly 60% and it will soon be entering phase 1 studies. To provide an improved predictability of this compound and its PD markers in the clinic we correlated PK, PD (phosphoproteins and FDG-PET imaging) and tumor growth inhibition in a single model. Since this inhibitor exhibits an IC50 of 37 nM in the PC3 prostate cell line (PTEN null) in vitro we chose this cell line for a xenograft model. Three groups (Vehicle, 5 and 7.5 mg/kg.) were dosed p.o. daily and subsequently evaluated. Tissues were harvested on day 7 and 13 at 4.5 hr post-dose. FDG-PET imaging was completed on days 1, 3, 7 and 13 at 3 hr post-dose. First, negative correlations were established between blood levels of PF-04691502 and the following tumor phosphoprotein epitopes: pS6, pAKTS473 and pAKTT308. The R2 values for these proteins were −0.3, −0.4 and −0.4, respectively. P-values of the correlations are all significant for all three proteins. Second, all PD markers were positively correlated with TGI. When correlating FDG-PET and TGI, day 7 has the best correlation and an R2 of 0.3 while days 3 and 13 have poorer correlations (R2 of 0.04 and 0.13, respectively). Although the R2 isn't high for day 7, the positive trend between image and TGI is statistically significant. When correlating PD markers and TGI, R2 values for pS6, pAKTS473 and pAKTT308 were 0.6, 0.8 and 0.6, respectively. In conclusion, the data from this single model indicates PD phosphoprotein markers are more reflective of TGI than FDG-PET imaging. These results will be applied to estimating the size of the cohort recruited for mechanistic evaluation in phase 1 trials of PF-04691502. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3492.

LPLI inhibits apoptosis upstream of Bax translocation via a GSK‐3β‐inactivation mechanism

Low-power laser irradiation (LPLI), a non-damage physical therapy, which has been used clinically for decades of years, is shown to promote cell proliferation and prevent apoptosis. However, the underlying mechanisms that LPLI prevents cell apoptosis remain undefined. In this study, based on real-time single-cell analysis, we demonstrated for the first time that LPLI inhibits staurosporine (STS)-induced cell apoptosis by inactivating the GSK-3beta/Bax pathway. LPLI could inhibit the activation of GSK-3beta, Bax, and caspase-3 induced by STS. In the searching for the mechanism, we found that, LPLI can activate Akt, which was consistence with our former research, even in the presence of STS. In this anti-apoptotic process, the interaction between Akt and GSK-3beta increased gradually, indicating Akt interacts with and inactivates GSK-3beta directly. Conversely, LPLI decreased the interaction between GSK-3beta and Bax, with the suppression of Bax translocation to mitochondria, suggesting LPLI inhibits Bax translocation through inactivating GSK-3beta. These results were further confirmed by the experiments of co-immunoprecipitation. Wortmannin, an inhibitor of phosphatidylinositol 3'-OH kinase (PI3K), potently suppressed the activation of Akt and subsequent anti-apoptotic processes induced by LPLI. Taken together, we conclude that LPLI protects against STS-induced apoptosis upstream of Bax translocation via the PI3K/Akt/GSK-3beta pathway. These findings raise the possibility of LPLI as a promising therapy for neuron-degeneration disease induced by GSK-3beta.

Fibrosis in Liver As A Predictive Marker for Hepatitis C Virus Therapy

We read with great interest the article by Petta et al.1 The compound 25-hydroxyvitamin D3 (25[OH]D3) was reported as an independent predictor of cardiovascular disease (by a decreased expression of profibrotic markers, and an increased expression of antifibrotic markers) despite the fact that its real pathological pathway is still not clear.2 Incubation of the multipotent mesenchymal cell with 25(OH)D3 also resulted in antiproliferative and antiapoptotic processes.2 Therefore, the lower levels of 25(OH)D3 in liver with greater fibrosis is understandable. Lower cholesterol and lower 25(OH)D3 levels, along with greater steatosis, were found to be risk factors affecting sustained virological response (SVR) as seen in recent studies. The stage of fibrosis was found to be a risk factor for SVR not only in hepatitis C virus (HCV) alone, but also in patients coinfected with human immunodeficiency virus and HCV, in contrast to the results of the current article.3, 4 Moreover, age, sex, and body mass index were also described as predictors for SVR in patients infected with HCV,5 in contrast to the current study. These challenging results could be related in the methodologic differences between the present study and recent studies, or mistakes could have happened during the sampling and/or analyzing periods. For example, SVR was reached in the half the male patients, whereas it was reached in just one-third of the females, results which are also different from the recent data. The patients in the study may also be infected with an unknown subgroup of HCV, which could explain these patients' characteristics. Akif Altınbaş M.D.*, Şahin Coban M.D.*, Osman Yüksel M.D.*, * Department of Gastroenterology, Dışkapı Yıldırım Beyazit Education and Research Hospital, Ankara, Turkey.