Ser81 Survivin Induced Protein Kinase A (PKA)-dependent Phosphatidylinositol 3-kinase (PI3K) Activity

BACKGROUND: Our previous report showed that phosphorylated-survivin at Ser81 induces survivin back loop to activate protein kinase A (PKA) in the cytoprotection mechanism. Activated PKA could possibly induce the cytoprotection via Phosphatydilinositol 3-kinase (PI3K). Therefore our current study was conducted to investigate the possibility of survivin-PKA-PI3K signaling pathway.METHODS: Viral productions by BOSC23 cells of Survivin, Antisense Survivin (Survivin-AS) and Ser81Ala mutant (Survivin-S81A) in pMSCV-IRES-GFP vector with cytomegalovirus (CMV) promoter were conducted. L929 cells were pretreated with/without PKI 6-22 amide and infected with viral particle of Survivin, Survivin-AS, Survivin-S81A or vector only. Cells were harvested, lysed and immunoprecipitated with anti-PI3K (p85) antibody and immunoblotted to detect PI3K (p85) and phospho-(Tyr) p85 PI3K. To confirm PI3K activation, PI3K Activity Assay was conducted by using phosphoinositide fraction containing PtdIns(4,5)P2 and [32P]ATP.RESULTS: Immunoblot and PI3K activity results showed similar results. Upon infection of virus with survivin, a markedly increased level of tyrosine phosphorylation of p85 PI3K or PI3K activity in L929 cells was seen. Low levels of tyrosine phosphorylation of p85 PI3K or PI3K activity were observed for Survivin-AS and Survivin-S81A-viral-infected L929 cells. With higher concentrations of Survivin-viral-infection, levels of tyrosine phosphorylation of p85 PI3K or PI3K activity in L929 cells were gradually increased. However, when L929 cells were pretreated with PKI 6-22 amide, prior to Survivin-viral-infection, level of tyrosine phosphorylation level of p85 PI3K or PI3K activity was detected much lower.CONCLUSION: Our result suggest that Ser81 Survivin play role in inducing PI3K activation and the Survivin-PI3K signaling pathway was PKA-dependent.KEYWORDS: Ser81, Survivin, PKA, PI3K, L92

Daxx, a Novel Fas-Binding Protein That Activates JNK and Apoptosis

The Fas cell surface receptor induces apoptosis upon receptor oligomerization. We have identified a novel signaling protein, termed Daxx, that binds specifically to the Fas death domain. Overexpression of Daxx enhances Fas-mediated apoptosis and activates the Jun N-terminal kinase (JNK) pathway. A C-terminal portion of Daxx interacts with the Fas death domain, while a different region activates both JNK and apoptosis. The Fas-binding domain of Daxx is a dominant-negative inhibitor of both Fas-induced apoptosis and JNK activation, while the FADD death domain partially inhibits death but not JNK activation. The Daxx apoptotic pathway is sensitive to both Bcl-2 and dominant-negative JNK pathway components and acts cooperatively with the FADD pathway. Thus, Daxx and FADD define two distinct apoptotic pathways downstream of Fas

Fully Supramolecular Polyrotaxanes as Biphase Drug Delivery Systems

Pseudopolyrotaxanes (PPR) consisting of α-cyclodextrin rings and polyethylene glycol axes with end thymine groups have been synthesized and characterized successfully. Fluorescein (Fl) as a model drug was conjugated to the hydroxyl functional groups of cyclodextrin rings of PPR via ester bonds and PPR-Fl as the primary drug delivery system was obtained. Finally PPR-Fl was capped by hydrogen bonds between end thymine groups and a suitable complementary molecule such as polycitric acid, citric acid, or adenine. The aim of this work was to control the release of the fluorescein-cyclodextrin (Fl-CD) conjugates, as the secondary drug delivery systems, from PPR-Fl by controlling the noncovalent interactions between stoppers and thymine end groups. It was found that the rate of release of the Fl-CD from PPR-Fl could be controlled by pH and the ratio of citric acid or adenine to the PPR-Fl

Changes in membrane lipids drive increased endocytosis following Fas ligation

Once activated, some surface receptors promote membrane movements that open new portals of endocytosis, in part to facilitate the internalization of their activated complexes. The prototypic death receptor Fas (CD95/Apo1) promotes a wave of enhanced endocytosis that induces a transient intermixing of endosomes with mitochondria in cells that require mitochondria to amplify death signaling. This initiates a global alteration in membrane traffic that originates from changes in key membrane lipids occurring in the endoplasmic reticulum (ER). We have focused the current study on specific lipid changes occurring early after Fas ligation. We analyzed the interaction between endosomes and mitochondria in Jurkat T cells by nanospray-Time-of-flight (ToF) Mass Spectrometry. Immediately after Fas ligation, we found a transient wave of lipid changes that drives a subpopulation of early endosomes to merge with mitochondria. The earliest event appears to be a decrease of phosphatidylcholine (PC), linked to a metabolic switch enhancing phosphatidylinositol (PI) and phosphoinositides, which are crucial for the formation of vacuolar membranes and endocytosis. Lipid changes occur independently of caspase activation and appear to be exacerbated by caspase inhibition. Conversely, inhibition or compensation of PC deficiency attenuates endocytosis, endosome-mitochondria mixing and the induction of cell death. Deficiency of receptor interacting protein, RIP, also limits the specific changes in membrane lipids that are induced by Fas activation, with parallel reduction of endocytosis. Thus, Fas activation rapidly changes the interconversion of PC and PI, which then drives enhanced endocytosis, thus likely propagating death signaling from the cell surface to mitochondria and other organelles

Prevalence of metabolic syndrome and its determinants among Iranian adults : evidence of IraPEN survey on a bi-ethnic population

Metabolic syndrome (MetS) is a growing public health concern worldwide. It has been demonstrated that individuals with MetS are at an increased risk of cardiovascular events and diabetes. We aimed to investigate the prevalence of MetS and its components among Turkic and Kurds ethnic groups in a bi-ethnic (Turk and Kurd) population. This cross-sectional study is part of the national health transformation plan created in response to the emerging epidemic of non-communicable diseases (Iran's Package of Essential Non-communicable Disease study), launched in 2014 in Naqadeh, Iran. In total, 3506 participants aged 30-70 years were randomly included in the study from urban and rural regions. Cardio-metabolic risk factors related to MetS diagnosis and other related sociodemographic factors were assessed for men and women in both the Turk and the Kurd population. Multivariate logistic regressions were applied to identify MetS-associated factors among both the Turk and the Kurd population. The mean (SD) age of the participants was 49.6 (12.3) years. Of the participants, 56.2% (n = 1969) were women, and 43.8% (n = 1537) were men. Three-fifths of the participants were Turk (60.3%, n = 1751). The overall prevalence of MetS was 37.05%, with a higher prevalence in women (49.8% versus 24.3% in men). The prevalence of MetS and its components among Turk people (41.6%) were significantly higher than that among Kurd people (33.9%) (p < 0.0001). In addition, the prevalence of MetS was higher among women, urban, and older people for both ethnicities. Strong associations were found between MetS prevalence and being older, being female, being overweight, being obese, having a higher waist-to-hip ratio, and having a history of diabetes and cardiovascular disease (CVD) in the family for both Turks and Kurds. The raised waist circumference (WC) is the most prevalent MetS component for Turk men and women. Meanwhile, the most prevalent MetS component for Kurd participants is low high-density lipoprotein for women and a raised WC for men. Significant differences were found between Kurdish men and women for all components, except for a raised WC and a raised fasting blood glucose (p < 0.05). Because the Iranian population features multiple ethnicities, the recognition of the prevalence of MetS components is a major step in establishing intervention strategies for modifying cardio-metabolic risk factors based on the population ethnicities and their sociodemographic, cultural, and lifestyle factors. We recommend future studies for planning an efficient and sustainable health education and promotion program to halt MetS prevalence

Transformation by Rho exchange factor oncogenes is mediated by activation of an integrin-dependent pathway.

Constitutive activation of growth factor receptor signaling pathways leads to uncontrolled growth, but why tumor cells become anchorage independent is less clear. The fact that integrins transmit signals required for cell growth suggests that constitutive activation of steps downstream from integrins mediates anchorage independence. Since the small GTPase Rho may mediate integrin signal transduction, the effects of serum and the Rho nucleotide exchange factor oncogenes dbl and lbc on cell growth and signaling pathways were examined. Our data show that these oncogenes induce anchorage-independent but serum-dependent growth and stimulation of signaling pathways. These results show, therefore, that anchorage-independent growth results from constitutive activation of integrin-dependent signaling events. They also support the view that Rho is a functionally important mediator of integrin signaling

Blocks to thyroid cancer cell apoptosis can be overcome by inhibition of the MAPK and PI3K/AKT pathways

Current treatment for recurrent and aggressive/anaplastic thyroid cancers is ineffective. Novel targeted therapies aimed at the inhibition of the mutated oncoprotein BRAFV600E have shown promise in vivo and in vitro but do not result in cellular apoptosis. TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a tumor-selective manner by activating the extrinsic apoptotic pathway. Here, we show that a TRAIL-R2 agonist antibody, lexatumumab, induces apoptosis effectively in some thyroid cancer cell lines (HTh-7, TPC-1 and BCPAP), while more aggressive anaplastic cell lines (8505c and SW1736) show resistance. Treatment of the most resistant cell line, 8505c, using lexatumumab in combination with the BRAFV600E inhibitor, PLX4720, and the PI3K inhibitor, LY294002, (triple-drug combination) sensitizes the cells by triggering both the extrinsic and intrinsic apoptotic pathways in vitro as well as 8505c orthotopic thyroid tumors in vivo. A decrease in anti-apoptotic proteins, pAkt, Bcl-xL, Mcl-1 and c-FLIP, coupled with an increase in the activator proteins, Bax and Bim, results in an increase in the Bax to Bcl-xL ratio that appears to be critical for sensitization and subsequent apoptosis of these resistant cells. Our results suggest that targeting the death receptor pathway in thyroid cancer can be a promising strategy for inducing apoptosis in thyroid cancer cells, although combination with other kinase inhibitors may be needed in some of the more aggressive tumors initially resistant to apoptosis

Combination of Bortezomib and Mitotic Inhibitors Down-Modulate Bcr-Abl and Efficiently Eliminates Tyrosine-Kinase Inhibitor Sensitive and Resistant Bcr-Abl-Positive Leukemic Cells

Emergence of resistance to Tyrosine-Kinase Inhibitors (TKIs), such as imatinib, dasatinib and nilotinib, in Chronic Myelogenous Leukemia (CML) demands new therapeutic strategies. We and others have previously established bortezomib, a selective proteasome inhibitor, as an important potential treatment in CML. Here we show that the combined regimens of bortezomib with mitotic inhibitors, such as the microtubule-stabilizing agent Paclitaxel and the PLK1 inhibitor BI2536, efficiently kill TKIs-resistant and -sensitive Bcr-Abl-positive leukemic cells. Combined treatment activates caspases 8, 9 and 3, which correlate with caspase-induced PARP cleavage. These effects are associated with a marked increase in activation of the stress-related MAP kinases p38MAPK and JNK. Interestingly, combined treatment induces a marked decrease in the total and phosphorylated Bcr-Abl protein levels, and inhibits signaling pathways downstream of Bcr-Abl: downregulation of STAT3 and STAT5 phosphorylation and/or total levels and a decrease in phosphorylation of the Bcr-Abl-associated proteins CrkL and Lyn. Moreover, we found that other mitotic inhibitors (Vincristine and Docetaxel), in combination with bortezomib, also suppress the Bcr-Abl-induced pro-survival signals and result in caspase 3 activation. These results open novel possibilities for the treatment of Bcr-Abl-positive leukemias, especially in the imatinib, dasatinib and nilotinib-resistant CML cases

The Mitogen-activated Protein Kinase Phosphatases PAC1, MKP-1, and MKP-2 Have Unique Substrate Specificities and Reduced Activity in Vivo toward the ERK2 sevenmaker Mutation

Mitogen-activated protein (MAP) kinases can be grouped into three structural families, ERK, JNK, and p38, which are thought to carry out unique functions within cells. We demonstrate that ERK, JNK, and p38 are activated by distinct combinations of stimuli in T cells that simulate full or partial activation through the T cell receptor. These kinases are regulated by reversible phosphorylation on Tyr and Thr, and the dual specific phosphatases PAC1 and MKP-1 previously have been implicated in the in vivo inactivation of ERK or of ERK and JNK, respectively. Here we characterize a new MAP kinase phosphatase, MKP-2, that is induced in human peripheral blood T cells with phorbol 12-myristate 13-acetate and is expressed in a variety of nonhematopoietic tissues as well. We show that the in vivo substrate specificities of individual phosphatases are unique. PAC1, MKP-2, and MKP-1 recognize ERK and p38, ERK and JNK, and ERK, p38, and JNK, respectively. Thus, individual MAP kinase phosphatases can differentially regulate the potential for cross-talk between the various MAP kinase pathways. A hyperactive allele of ERK2 (D319N), analogous to the Drosophila sevenmaker gain-of-function mutation, has significantly reduced sensitivity to all three MAP kinase phosphatases in vivo

c-Fos as a Proapoptotic Agent in TRAIL-Induced Apoptosis in Prostate Cancer Cells

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)/Apo-2L promotes apoptosis in cancer cells while sparing normal cells. Although many cancers are sensitive to TRAIL-induced apoptosis, some evade the proapoptotic effects of TRAIL. Therefore, differentiating molecular mechanisms that distinguish between TRAIL-sensitive and TRAIL-resistant tumors are essential for effective cancer therapies. Here, we show that c-Fos functions as a proapoptotic agent by repressing the antiapoptotic molecule c-FLIP(L). c-Fos binds the c-FLIP(L) promoter, represses its transcriptional activity, and reduces c-FLIP(L) mRNA and protein levels. Therefore, c-Fos is a key regulator of c-FLIP(L), and activation of c-Fos determines whether a cancer cell will undergo cell death after TRAIL treatment. Strategies to activate c-Fos or inhibit c-FLIP(L) may potentiate TRAILbased proapoptotic therapies