Serum Deprivation-induced Apoptosis Research Articles

Over-expression of the X-linked inhibitor of apoptosis protein (XIAP) delays serum deprivation-induced apoptosis in CHO-K1 cells

Serum deprivation inhibits cell growth and initiates apoptosis cell death in mammalian cell cultures. Since apoptosis is a genetically controlled cell death pathway, over-expression of anti-apoptotic proteins may provide a way to delay apoptosis. This study investigated the ability of the X-linked inhibitor of apoptosis protein (XIAP) to inhibit apoptosis induced by serum deprivation. Study includes evaluation of the ability of XIAP to prolong culture period and its effect on cell proliferation in serum-deprived media. The full length human XIAP was introduced into CHO-K1 cell lines and the effects of XIAP over-expression on the inhibition of apoptosis induced by serum-deprived conditions were examined. In batch cultures, cells over-expressing XIAP showed decreased levels of apoptosis and a higher number of viable cell under serum-deprived conditions compared to the control cell lines. The viability of control cells dropped to 40% after 2days of serum deprivation, the XIAP expressing cells still maintained at a viability higher than 90%. Further investigation revealed that the caspase-3 activity of the CHO-K1 cell line was inhibited as a result of XIAP expression.

Bilobalide prevents apoptosis through activation of the PI3K/Akt pathway in SH-SY5Y cells

Bilobalide, a sesquiterpene trilactone constituent of Ginkgo biloba leaf extracts, has been proposed to exert protective and trophic effects on neurons. However, mechanisms underlying the protective effects of bilobalide remain unclear. Using human SH-SY5Y neuroblastoma cells and primary hippocampal neurons, this study investigated the neuroprotective effects of bilobalide. We mimicked aging-associated neuronal impairments by applying external factors (beta amyloid protein (Abeta) 1-42, H(2)O(2) and serum deprivation) consequently inducing cell apoptosis. As markers for apoptosis, cell viability, DNA fragmentation, mitochondrial membrane potential and levels of cleaved caspase 3 were measured. We found that, bilobalide prevented Abeta 1-42-, H(2)O(2)- and serum deprivation-induced apoptosis. To better understand the neuroprotective effects of bilobalide, we also tested the ability of bilobalide to modulate pro-survival signaling pathways such as protein kinase C (PKC), extracellular-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. It was found that, bilobalide dose-dependently increased PI3K activity and levels of phosphorylated Akt (p-Akt Ser473 and Thr308), which could be maintained up to at least 2 h after bilobalide withdrawal in cells treated with or without Abeta 1-42, H(2)O(2) or serum-free medium. In addition, application of PI3K/Akt inhibitor LY294002 could abrogate both the protective effects of bilobalide against Abeta 1-42-, H(2)O(2)- and serum deprivation-induced apoptotic cell damage and bilobalide-induced increase in PI3K activity and levels of p-Akt (Ser473 and Thr308). In contrast, application of PKC inhibitor staurosporine (STS) did not affect the protective effects of bilobalide. Moreover, no change in levels of phosphorylated ERK1/2 (p-ERK1/2) was observed in bilobalide-treated cells. These results further suggested that the PI3K/Akt pathway might be involved in the protective effects of bilobalide. Since modern technology allows production of purified bilobalide with high bioavailability, bilobalide may be useful in developing therapy for diseases involving age-associated neurodegeneration.

Proteinase‐activated receptor‐2 (PAR2) and mouse osteoblasts: Regulation of cell function and lack of specificity of PAR2‐activating peptides

1. Using synthetic proteinase-activated receptor-2 (PAR(2))-activating peptides (PAR(2)APs) corresponding to the tethered ligand domain of the extracellular N-terminus of PAR(2) to mimic the actions of activating proteinases and using primary cultures of calvarial osteoblasts derived from both wild-type (WT) and PAR(2)-null (KO) mice, we investigated the potential role of PAR(2) in regulating osteoblast function. 2. Primary calvarial osteoblasts from WT and KO mice were evaluated for their growth kinetics and mineralization in the absence of PAR(2) agonists and for their responses in a variety of functional assays to the PAR(2)APs Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-NH(2)) and 2-furoyl-Leu-Ile-Gly-Arg-Leu-Orn-amide (2-fLIGRLO-NH(2)), as well as to trypsin. 3. In contrast with WT cells, PAR(2)-KO osteoblasts did not exhibit increased collagen Type I mRNA expression in response to SLIGRL-NH(2). When grown in serum-containing medium, KO cells increased in number more rapidly than WT cells, an effect that could be attributed to decreased apoptosis rather than increased proliferation. Surprisingly, in both WT and KO osteoblasts, the two PAR(2)APs induced mobilization of intracellular calcium stores. Similarly, the PAR(2)APs inhibited serum deprivation-induced apoptosis and parathyroid hormone-, 1,25-dihydroxyvitamin D(3)- or interleukin-11-induced mineralization in WT and KO cells. 4. We conclude that PAR(2) plays a role in osteoblast survival and collagen Type I mRNA induction and that osteoblasts can respond to the PAR(2)APs via both PAR(2)-dependent and -independent mechanisms.

Neural-specific Deletion of FIP200 Leads to Cerebellar Degeneration Caused by Increased Neuronal Death and Axon Degeneration

FIP200 (FAK family-interacting protein of 200 kDa) is a conserved protein recently identified as a potential mammalian counterpart of yeast autophagy protein Atg17. However, it remains unknown whether mammalian FIP200 regulates autophagy in vivo. Here we show that neural-specific deletion of FIP200 resulted in cerebellar degeneration accompanied by progressive neuronal loss, spongiosis, and neurite degeneration in the cerebellum. Furthermore, deletion of FIP200 led to increased apoptosis in cerebellum as well as accumulation of ubiquitinated protein aggregates without any deficiency in proteasome catalytic functions. We also observed an increased p62/SQSTM1 accumulation in the cerebellum and reduced autophagosome formation as well as accumulation of damaged mitochondria in the mutant mice. Lastly, analysis of cerebellar neurons in vitro showed reduced JNK activation and increased susceptibility to serum deprivation-induced apoptosis in cerebellar neurons from the mutant mice. Taken together, these results provide strong genetic evidence for a role of FIP200 in the regulation of neuronal homeostasis through its function in autophagy in vivo.

Overexpressing cellular repressor of E1A-stimulated genes protects mesenchymal stem cells against hypoxia- and serum deprivation-induced apoptosis by activation of PI3K/Akt

Bone marrow-derived mesenchymal stem cells (MSCs) have great potential for repair after myocardial infarction. However, poor viability of transplanted MSCs in the ischemic heart has limited their therapeutic potential. Cellular repressor of E1A-stimulated genes (CREG) has been identified as a potent inhibitor of apoptosis. The aim of this study was to investigate the anti-apoptotic effects of CREG on MSCs under hypoxic and serum deprivation (SD) conditions. We also investigated the potential mechanism(s) that may mediate the actions of CREG. All experiments were performed on rat bone marrow MSCs. Apoptosis was induced by exposure of cells to hypoxia/SD in a sealed GENbox hypoxic chamber. Effects of CREG were investigated in the absence or presence of inhibitors that target phosphoinositide 3-kinase (PI3K). We found that the overexpression of CREG markedly protected MSCs from hypoxia/SD-induced apoptosis through inhibition of the mitochondrial apoptotic pathway, leading to attenuation of caspase-3. Moreover, CREG enhanced Akt phosphorylation and decreased the expression of p53 in MSCs under hypoxic/SD conditions. The PI3K/Akt inhibitor LY294002 significantly increased the amount of p53 protein and attenuated the anti-apoptotic effects of CREG on MSCs. This study indicates that CREG is a novel and potent survival factor for MSCs, therefore, it may be a useful therapeutic adjunct for transplanting MSCs into damaged heart after myocardial infarction.

Serum deprivation-induced reactive oxygen species production is mediated by Romo1

Serum deprivation-triggered increases in reactive oxygen species (ROS) are known to induce apoptotic cell death. However, the mechanism by which serum deprivation causes ROS production is not known. Since mitochondria are the main source of ROS and since mitochondrial ROS modulator 1 (Romo1) is involved in ROS production, we sought to determine if serum deprivation triggered ROS production through Romo1. To examine the relationship between Romo1 and the serum deprivation-triggered increase in ROS, we transfected Romo1 siRNA into various cell lines and looked for inhibition of mitochondrial ROS generation. Romo1 knockdown by Romo1 siRNA blocked the mitochondrial ROS production caused by serum deprivation, which originates in the mitochondrial electron transport chain. We also found that Romo1 knockdown inhibited serum deprivation-induced apoptosis. These findings suggest that Romo1-derived ROS play an important role in apoptotic cell death triggered by withdrawal of cell survival factors.

Abstract 5556: Immunoglobulin-like Cell Adhesion Molecule Necl-5 Regulates Vascular Endothelial Growth Factor-Induced Angiogenesis by Controlling the Interaction Between VEGFR2 and Integrin α v β 3

Background Peripheral arterial disease (PAD) is highly prevalent worldwide. PAD is a major cause of lower limb amputation, which is associated with a poor quality of life and prognosis. Vascular endothelial growth factor (VEGF) is a key pro-angiogenic cytokine proposed as therapeutic angiogenesis for PAD. However, more investigation is needed to better understand VEGF-induced angiogenesis and its application to PAD. The activity of VEGF receptor 2 (VEGFR2) is regulated by direct interactions with VEGF and other cell surface proteins, including integrin α v β 3. However, the regulation mechanism of VEGFR2 and integrin α v β 3 interaction has not been established. Nectin-like molecules (Necls) are immunoglobulin-like cell adhesion molecules (CAMs) that regulate cell movement, proliferation and survival. Here we show that Necl-5, a member of Necls that binds integrin α v β 3, regulates the interaction between VEGFR2 and integrin α v β 3 in vascular endothelial cells (ECs) and that this CAM plays an important role in angiogenesis. Methods and Results Immunohistochemistry revealed that Necl-5 was preferentially expressed in ECs including mouse hind limb capillary endothelium. Flow cytometry showed that Necl-5 was expressed in various cultured ECs. Although Necl-5-knockout mice displayed no obvious defect in vascular development, these mice exhibited impaired adaptive angiogenesis following hind limb ischemia. To clarify the regulation mechanism of angiogenesis by Necl-5, we knocked down Necl-5 by siRNA and studied the role of Necl-5 in angiogenesis in vitro . VEGF-induced tube formation on Matrigel, migration, and proliferation were significantly inhibited in Necl-5-knockdown ECs. Conversely, serum deprivation-induced apoptosis was enhanced in Necl-5-knockdown ECs. Knockdown of Necl-5 prevented VEGF-induced interaction of integrin α v β 3 with VEGFR2, and then suppressed the VEGFR2-mediated activation of Akt, a critical signal for angiogenesis and cell survival. Conclusions These findings indicate that Necl-5 regulates VEGF-induced angiogenesis by controlling the interaction between VEGFR2 and integrin α v β 3, and the VEGFR2-mediated signaling. Therapeutic intervention of Necl-5 may represent a novel strategy in the patients with PAD.

Abstract 5592: Role of Afadin in Vascular Endothelial Growth Factor- and Sphingosine 1-Phosphate-Induced Angiogenesis

[Background] Better understanding of mechanism of angiogenesis and angiogenic signaling of pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate (S1P) will aid in developing therapeutic angiogenesis for ischemic heart disease and peripheral arterial disease. Recently, Rap1 small G protein was identified as a key angiogenic signaling molecule. However, mechanisms involved in the Rap1 regulation of angiogenesis and downstream effectors of Rap1 are not known. Afadin is an adherens junction protein that connects immunoglobulin-like adhesion molecule nectins to the actin cytoskeleton and binds activated Rap1. Recently, we identified that, besides the role in cell-cell adhesion formation in epithelial cells, afadin plays a role in the regulation of directional cell movement of fibroblasts. We therefore attempted to investigate the role of endothelial afadin in angiogenesis and clarify the underlying molecular mechanism. [Method and Results] Knockdown of Rap1 or afadin by siRNA inhibited the VEGF- and S1P-induced tubulogenesis, migration and proliferation, and conversely, increased the serum deprivation-induced apoptosis of human umbilical vein endothelial cells (HUVECs). Immunoprecipitation and immunofluorescence studies revealed that Rap1 regulated the interaction between afadin and phosphatidyl-inositol 3-kinase (PI3K) and recruitment of the afadin-PI3K complex to the leading edge, suggesting that Rap1 and afadin regulates the PI3K signaling. Indeed, knockdown of Rap1 or afadin by siRNA inhibited the VEGF- and S1P-induced phosphorylation of Akt. Thus, afadin regulated the PI3K-Akt signaling. To examine the role of afadin in angiogenesis in vivo, we developed endothelial cell-specific afadin-knockout mice using the Cre/loxP system. Majority (96%) of afadin-knockout mice were embryonically lethal, suggesting an essential role of endothelial afadin in embryonic vascular development and angiogenesis. We performed Matrigel plug assay in born adult afadin-knockout mice and observed that VEGF- and S1P-induced angiogenesis was impaired in afadin-knockout mice. [Conclusion] These results demonstrate that afadin regulates VEGF- and S1P-induced angiogenesis downstream of Rap1.

Novel Dehydroepiandrosterone Derivatives with Antiapoptotic, Neuroprotective Activity

DHEA analogues with modifications at positions C3 or C17 were synthesized and evaluated for neuroprotective activity against the neural-crest-derived PC12 cell model of serum deprivation-induced apoptosis. The most potent compounds were the spiro-epoxy derivatives 17beta-spiro[5-androstene-17,2'-oxiran]-3beta-ol (20), (20S)-3beta,21-dihydroxy-17beta,20-epoxy-5-pregnene (23), and (20R)-3beta,21-dihydroxy-17alpha,20-epoxy-5-pregnene (27) with IC(50) values of 0.19 +/- 0.01, 99.0 +/- 4.6, and 6.4 +/- 0.3 nM, respectively. Analogues 20, 23, and 27, up to the micromolar range of concentrations, were unable to activate estrogen receptor alpha and beta (ERalpha and ERbeta) or to interfere with ER-dependent gene expression significantly. In addition, they were unable to stimulate the growth of Ishikawa, MCF-7, and LNCaP cells. Our results suggest that the spiro-epoxyneurosteroid derivatives 20, 23, and 27 may prove to be lead molecules for the synthesis of novel neuroprotective agents.

MTORC1 Hyperactivity Inhibits Serum Deprivation-Induced Apoptosis via Increased Hexokinase II and GLUT1 Expression, Sustained Mcl-1 Expression, and Glycogen Synthase Kinase 3β Inhibition

The current concept is that Tsc-deficient cells are sensitized to apoptosis due to the inhibition of Akt activity by the negative feedback mechanism induced by the hyperactive mTORC1. Unexpectedly, however, we found that Tsc1/2-deficient cells exhibit increased resistance to serum deprivation-induced apoptosis. mTORC1 hyperactivity contributes to the apoptotic resistance of serum-deprived Tsc1/2-deficient cells in part by increasing the growth factor-independent expression of hexokinase II (HKII) and GLUT1. mTORC1-mediated increase in hypoxia-inducible factor 1alpha (HIF1alpha) abundance, which occurs in the absence of serum in normoxic Tsc2-deficient cells, contributes to these changes. Increased HIF1alpha abundance in these cells is attributed to both an increased level and the sustained translation of HIF1alpha mRNA. Sustained glycogen synthase kinase 3beta inhibition and Mcl-1 expression also contribute to the apoptotic resistance of Tsc2-deficient cells to serum deprivation. The inhibition of mTORC1 activity by either rapamycin or Raptor knockdown cannot resensitize these cells to serum deprivation-induced apoptosis because of elevated Akt activity that is an indirect consequence of mTORC1 inhibition. However, the increased HIF1alpha abundance and the maintenance of Mcl-1 protein expression in serum-deprived Tsc2(-/)(-) cells are dependent largely on the hyperactive eIF4E in these cells. Consistently, the reduction of eIF4E levels abrogates the resistance of Tsc2(-/)(-) cells to serum deprivation-induced apoptosis.

Antiapoptotic effects of vasopressin in the neuronal cell line H32 involve protein kinase Cα and β

Activation of V1 vasopressin (VP) receptors prevents serum deprivation-induced apoptosis in neuronal H32 cells, partially through mitogen-activated protein kinase (MAPK) mediated Bad phosphorylation. In this study, we investigated the role of protein kinases C (PKC) and B (PKB) mediating VP-induced antiapoptosis in H32 cells. Serum deprivation increased PKCdelta but not PKCalpha or PKCbeta activity, while VP increased PKCalpha and PKCbeta without affecting PKCdelta activity. Inhibition of PKCdelta prevented caspase 3 activation, indicating that PKCdelta mediates the pro-apoptotic actions of serum deprivation. Simultaneous inhibition of PKCalpha and beta and MAPK abolished VP-induced Bad phosphorylation, but it only partially prevented caspase 3 inhibition. Complete abolition of the protective effect of VP on serum deprivation-induced caspase 3 activity required additional blockade of phosphoinositide 3 kinase (PI3K)/protein kinase B. The data demonstrate that VP exerts antiapoptosis through multiple pathways; while PKCalpha and beta together with extracellular signal-regulated kinases/MAPK activation mediates Bad phosphorylation (inactivation), the full protective action of VP requires additional activation of PKB (PI3K/protein kinase B) pathway.

Mechanisms of Panax ginseng in preventing rat pheochromocytoma cells from apoptosis

Aims of the study Although ginseng root possesses dominant central therapeutic effects and has recently undergone investigations for treating different neuronal diseases, most of its mechanisms are still unknown. Therefore, the neuroprotective mechanisms of ginseng were studied. Materials and methods The protection afforded by different methanol extracts of Panax ginseng (PG) was tested in a serum deprivation-induced apoptotic model using neuronal-like pheochromocytoma (PC12) cells. An MTT assay, annexin V-FITC staining, and Western blots were, respectively, applied to identify the viability of cells, the apoptotic form of cell death, and the activity of antiapoptotic signaling. Results The known antiapoptotic PI3-K/Akt and MEK/ERK pathways in this system were ruled out due to failure of LY 294002 and PD 98059 to block the protection by PG. A protein kinase A (PKA) inhibitor was found to block the protection by PG and PG-induced CREB phosphorylation, suggesting that the PKA/CREB pathway mediates the protective effect of PG. Downregulation of classical and novel PKCs failed to block the protection by PG, while an atypical PKC inhibitor blocked protection by PG. Conclusions PKA and atypical PKC are important for the protection afforded by PG in preventing serum deprivation-induced PC12 cell apoptosis.

Adipose stromal cells-secreted neuroprotective media against neuronal apoptosis

Transplantation of pluripotent adipose stem/stromal cells (ASC) alleviates tissue damage and improves functional deficits in both stroke and cardiovascular disease animal models. Recent studies indicate that the primary mechanism of ASC-induced repair may not be directly related to tissue regeneration through differentiation, but rather through paracrine mechanisms provided by secreted pro-survival and repair-inducing trophic factors. In this study, we have found that ASC-conditioned medium (ASC-CM) potently protected cerebellar granule neurons (CGN) from apoptosis induced by serum and potassium deprivation. Neural cell protection was mostly attributable to activated caspase-3 and Akt-mediated neuroprotective pathway signaling. Specific neutralization of neurotrophic factor activity demonstrated that serum and potassium deprivation-induced Akt-mediated neuroprotection and caspase-3-dependent apoptosis were mainly modulated by IGF-1. These data suggest that of the many neuroprotective factors secreted by ASC, IGF-1 is the major factor that mediates protection against serum and potassium deprivation-induced CGN apoptosis. This study establishes a mechanistic basis supporting the therapeutic application of ASC for neurological disorders, specifically through paracrine support provided by trophic factor secretion.

Transforming growth factor-β1 causes pulmonary microvascular endothelial cell apoptosis via ALK5

We have previously shown that transforming growth factor (TGF)-beta1 protected against main pulmonary artery endothelial cell (PAEC) apoptosis induced by serum deprivation and VEGF receptor blockade through a mechanism associated with ALK5-mediated Bcl-2 upregulation. In the current study, we investigated the effect of TGF-beta1 on pulmonary microvascular endothelial cell (PMVEC) apoptosis. We found that, in contrast to the results seen in conduit PAEC, TGF-beta1 caused apoptosis of PMVEC, an effect that was also dependent on ALK5 activity. We noted that non-SMAD signaling pathways did not play a role in TGF-beta1-induced apoptosis. Both SMAD2 and SMAD1/5 were activated upon exposure to TGF-beta1. TGF-beta1-induced activation of SMAD2, but not SMAD1/5, was abolished by ALK5 inhibition, an effect that associated with prevention of TGF-beta1-induced apoptosis. These results suggest that SMAD2 is important in TGF-beta1-induced apoptosis of PMVEC. While caspase-12 activity was not altered, caspase-8 was activated by TGF-beta1, an effect that correlated with a reduction of cFLIP protein levels. Additionally, TGF-beta1 decreased Bcl-2 protein levels and induced cytochrome c cytosolic redistribution. These results suggest that TGF-beta1 caused apoptosis of PMVEC likely through both caspase-8-dependent extrinsic pathway and mitochondria-mediated intrinsic pathway. We noted that inhibition of ALK5 attenuated serum deprivation-induced apoptosis, an effect that correlated with increased expression and activation of CREB and its potential target genes, Bcl-2 and cFLIP. These results suggest that CREB may be important in mediating apoptosis resistance of PMVEC upon ALK5 inhibition perhaps through upregulation of Bcl-2 and cFLIP. Finally, we noted that SMAD1/5 were activated upon ALK5 inhibition in the presence of low levels of TGF-beta1, an effect associated with enhanced endothelial proliferation. We speculate that imbalance of ALK1 and ALK5 may contribute to the development of pulmonary artery hypertension.

Regulation of heme oxygenase-1 expression and MAPK pathways in response to kaempferol and rhamnocitrin in PC12 cells

Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities, especially neural diseases. Our aim of research is to investigate the protective effects and mechanisms of kaempferol and rhamnocitrin (kaempferol-7-methyl ether) on oxidative damage in rat pheochromocytoma PC12 cells induced by a limited supply of serum and hydrogen peroxide (H2O2). The current result demonstrated that kaempferol protected PC12 cells from serum deprivation-induced apoptosis. Pretreatment of cells with kaempferol also diminished intracellular generation of reactive oxygen species (ROS) in response to H2O2 and strongly elevated cell viability. RT-Q-PCR and Western blotting revealed that kaempferol and rhamnocitrin significantly induced heme oxygenase (HO)-1 gene expression. Addition of zinc protoporphyrin (Znpp), a HO-1 competitive inhibitor, significantly attenuated their protective effects in H2O2-treated cells, indicating the vital role of HO-1 in cell resistance to oxidative injury. While investigating the signaling pathways responsible for HO-1 induction, we observed that kaempferol induced sustained extracellular signal-regulated protein kinase 1/2 (ERK1/2) in PC12 cells grown in low serum medium; while rhamnocitrin only stimulated transient ERK cascade. Addition of U0126, a highly selective inhibitor of MEK1/2, which is upstream of ERK1/2, had no effect on kaempferol- or rhamnocitrin-induced HO-1 mRNA expression, indicating no direct cross-talk between these two pathways. Furthermore, both kaempferol and rhamnocitrin were able to persistently attenuate p38 phosphorylation. Taking together, the above findings suggest that kaempferol and rhamnocitrin can augment cellular antioxidant defense capacity, at least in part, through regulation of HO-1 expression and MAPK signal transduction.

Ligusticum chuanxiong as a potential neuroprotectant for preventing serum deprivation-induced apoptosis in rat pheochromocytoma cells: Functional roles of mitogen-activated protein kinases

Ethnopharmacological relevance Ligusticum chuanxiong (LC) as a common component in many traditional Chinese medicinal formulas and decoctions has been used to treat different central nervous diseases, suggesting a neuroprotective function. Aim of the study To investigate the functional roles of mitogen-activated protein kinases (MAPKs) in mediating the neuroprotection of LC. Materials and methods Different extractions of LC were applied with or without MAPK inhibitor to test their protection against serum deprivation-induced apoptosis in rat neuronal-like pheochromocytoma (PC12) cells as revealed by an MTT assay or Hoechst staining. Western blot was used to identify the activations of MAPKs. Results The most effective butanol extraction (LC-BuOH) was used in the following experiments. LC-BuOH reversed serum deprivation-induced decreased phosphorylation of extracellular signal-regulated kinase (ERK) and increased phosphorylation of c-Jun NH 2-terminal kinase (JNK) and p38, the family of MAPKs. A PKA inhibitor, blocked the protection of LC-BuOH and partially blocked LC-BuOH-induced alterations in MAPKs, suggesting that the LC-BuOH regulates MAPKs through both PKA-dependent and -independent pathways. Although PD 98059, an inhibitor of MEK which activates ERK, blocked LC-BuOH-induced ERK phosphorylation, it did not block the protection of LC-BuOH. Conclusions LC-BuOH mediates protection by suppressing JNK/p38 instead of activating ERK activity.

Angiopoietin-1 Protects Endothelial Cells From Hypoxia-Induced Apoptosis via Inhibition of Phosphatase and Tensin Homologue Deleted From Chromosome Ten

Background and Objectives: Angiopoietin-1 (Ang1) is a regulator of blood vessel growth and maturation, and prevents radiation-induced or serum deprivation-induced apoptosis. Phosphatase and tensin homologue deleted from chromosome ten (PTEN), a well-known tumor suppressor, regulates cell cycle arrest and apoptosis. Hypoxia induces apoptosis by increasing the expression of PTEN. We hypothesized that Ang1 may regulate PTEN expression and, thus, reduce endothelial apoptosis under hypoxia in vitro and in vivo. Materials and Methods: In vitro, human umbilical vein endothelial cells (HUVECs) were treated with Ang1, and signaling pathways were investigated. In vivo, eight-week-old C57BL/6 mice were used for a hind limb ischemia model. Ang1 or normal saline was intramusculary injected. Blood flow was evaluated by a laser Doppler perfusion analyzer and tissue histology. Results: The expression of PTEN was markedly upregulated in HUVECs after hypoxic stimulation, whereas Ang1 suppressed PTEN expression. Tie2-Fc, a soluble form of Tie2 (sTie2) that blocks Ang1, reversed the Ang1 effect on PTEN reduction under hypoxia. Ang1 inhibited the nuclear translocation of nuclear transcription factor-kB (NFkB), a binding factor for the PTEN promoter and Foxo1. Hypoxia-induced p27 expression and apoptosis were also suppressed by Ang1. In the mouse hind limb ischemia model, we observed a high capillary density, numerous proliferating cells and diminished cell death in skeletal muscle tissue in the Ang1 injected group. Conclusion: Ang1 enhanced endothelial cell survival by reducing apoptosis via PTEN down-regulation in HUVECs under hypoxia. Local injection of Ang1 significantly reduced apoptotic cells in vivo, and prevented limb loss for ischemic hind limb mice. Thus, Ang1 may be an effective therapeutic for protection from ischemic-endothelial cell injury. (Korean Circ J 2009;39:57-65)

Protection against serum deprivation-induced apoptosis by docosahexaenoic acid in PC12 cells

Protection against serum deprivation-induced apoptosis by docosahexaenoic acid in PC12 cells

Characterization and biological functions of sulfated polysaccharides from sulfated-salt treatment of Antrodia cinnamomea

Sulfated polysaccharides (SPSs) of Antrodia cinnamomea were extracted by an exhaustive papain digestion and characterized, and their biological functions were evaluated. In this study, we demonstrated the existence of SPSs in the medicinal fungus, A. cinnamomea. Since no SPSs had previously been identified in any fungal organism, we attempted to characterize those from A. cinnamomea. SPSs from A. cinnamomea inhibited in vitro Matrigel tube formation, in an angiogenesis model, in a dose-dependent manner. Furthermore, using serum deprivation-induced apoptosis in neuronal-like PC12 cells as a stress model, the SPSs of A. cinnamomea were effective in preventing serum-deprived apoptosis. Compositional analysis revealed that myo-inositol, fucose, galactose, and glucose were the neutral sugars in SPSs of A. cinnamomea, and these SPSs had a high sulfate content. The sulfation degree paralleled their antiangiogenic and neuroprotective activities. In this work, we report novel data on the structure, antiangiogenic, and neuroprotective effects of these fungal SPSs.

Knockdown of human bid gene expression enhances survival of CD8 + T cells

Tumor cells have developed immune evasion mechanisms such as considerably heterogenous FasL expression on their surface via which they could induce apoptosis of tumor-specific cytotoxic T lymphocytes (CTLs) in the immune system. Meanwhile, the competition of normal immune cells with tumor cells results in relative growth factors shortage for growth and proliferation of nontumor cells, which improves a susceptibility to early apoptosis of CTL. In an attempt to develop strategies for prolonging the survival of adoptively transferred T cells in a hostile pro-apoptotic tumor microenvironment, we used synthetic siRNA and vector-based shRNA to suppress the expression of Bid in human uterocervical carcinoma HeLa cells, followed by the further achievement of Bid gene silencing in human primary cells-CD8 + lymphocytes via retrovirus-delivered siRNAs. Our results indicated that Bid knockdown HeLa cells are partially resistant to Fas antibody- or serum deprivation-induced apoptosis. Additionally, the blockade of Bid expression in CD8 + lymphocytes resulted in a less susceptiveness to Fas antibody-induced apoptosis and a survival advantage following recombinant human interleukin-2 (rhIL-2) withdrawal or under lower rhIL-2 concentrations compared with control lymphocytes . These data suggest that knockdown of Bid might serve as an approach to enhancing the survival and tumoricidal activity of T lymphocytes in adoptive immunotherapy.