Human papilloma virus type 16 E7 genes protect astrocytes against apoptotic and necrotic death induced by hydrogen peroxide.

Hydrogen peroxide is considered to be a dose- and time-dependent mediator in apoptotic and necrotic death. In this study, we examined the signaling of the E6 and E7 proteins with respect to apoptosis or necrosis after H2O2 injury using an in vitro model with overexpressed E6 or E7 genes. For this purpose, the E6 and E7 gene expressing astrocytes were exposed to 10 micromole and 200 micromole H2O2 solutions. Twenty- four hours after treatment with the lower dosage(10 micromole H2O2), control, E6-expressing cells suffered about 45% injury and LXSN-expressing cells decreased by 67% as assessed by LDH release. However, E7-expressing cells showed less injury, resulting in 20-30% of LDH release. Astrocytes expressing E6, E7, LXSN and mock-infected cells showed a typical apoptotic death pattern on the DNA gel after treatment with a low-dose of H2O2 (10 micromole), however they died from necrotic death after a high-dose (200 micromole) H2O2. Overexpression of HPV-E7 genes protected the cells from apoptotic death after a low-dose of H2O2 and from necrotic death after a high-dose of H2O2, while the overexpression of E6 genes from the necrotic death. E7 expressing astrocytes showed higher catalase activity and the levels of E2F protein surged more than 100-folds compared with the control astrocytes. We believe that the activity of E7 protein to protect astrocytes from H2O2 injury was at least partly due to increased catalase, a scavenger protein.     

Ghrelin protects against cell death of hippocampal neurons in pilocarpine-induced seizures in rats

Ghrelin, a 28-amino-acid peptide, is mainly secreted by the stomach. Evidence has shown ghrelin to have neuroprotective effects. However, whether ghrelin protects hippocampal neurons against cell death in pilocarpine-induced seizures is unknown. We used Nissl staining to show that ghrelin attenuated the neuronal loss caused by pilocarpine-induced seizures in the hippocampus. Ghrelin exerted the protective action through regulating the phosphatidylinositol-3-kinase and Akt pathway. Moreover, ghrelin treatment reversed the decreased ratio of Bcl-2 to Bax induced by seizures while inhibiting the activated caspase-3. Ghrelin can inhibit hippocampal neuronal damage caused by pilocarpine-induced seizures, which might have therapeutic value in seizures.     

Adenosine receptor ligands protect against a combination of apoptotic and necrotic cell death in cerebellar granule neurons

Agonists at A₁ receptors and antagonists at A_2A receptors are known to be neuroprotective against excitotoxicity. We set out to clarify the mechanisms involved by studying interactions between adenosine receptor ligands and endogenous glutamate in cultures of rat cerebellar granule neurons (CGNs). Glutamate and the selective agonist N-methyl-d-aspartate (NMDA), applied to CGNs at 9 div (days in vitro), both induced cell death in a concentration-dependent manner, which was attenuated by treatment with the NMDA receptor antagonists dizocilpine, d-2-amino-5-phosphono-pentanoic acid (d-AP5) or kynurenic acid (KYA), but not by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Glutamate toxicity was reduced in the presence of all of the following: cyclosporin A (CsA), a blocker of the membrane permeability transition pore, the caspase-3 inhibitor, benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone (Z-DEVD-fmk), the poly (ADP-ribose) polymerase (PARP-1) inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone (DPQ), and nicotinamide. This is indicative of involvement of both apoptotic and necrotic processes. The A₁ receptor agonist, N ⁶-cyclopentyladenosine (CPA), and the A_2A receptor antagonist 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazo-5-yl-amino]ethyl)phenol (ZM241385) afforded significant protection, while the A₁ receptor blocker 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and the A_2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxyamidoadenosine (CGS21680) had no effect. These results confirm that glutamate-induced neurotoxicity in CGNs is mainly via the NMDA receptor, but show that a form of cell death which exhibits aspects of both apoptosis and necrosis is involved. The protective activity of A₁ receptor activation or A_2A receptor blockade occurs against this mixed profile of cell death, and appears not to involve the selective inhibition of classical apoptotic or necrotic cascades.     

Chitooligosaccharides protect cultured hippocampal neurons against glutamate-induced neurotoxicity

Chitooligosaccharides (COSs), the biodegradation product of chitosan, have demonstrated a diverse array of biological activities. Here we report the protective effect of COSs (M.W. 800) against glutamate-induced neurotoxicity in cultured hippocampal neurons. The cell viability assessments, together with Hoechst 33342 staining and flow cytometry for cell apoptosis analysis, indicated that glutamate (125 μM)-induced cell apoptosis in cultured hippocampal neurons was attenuated in a concentration-dependent manner by COSs pretreatment. After measurement with Fluo 4-AM, COSs were found to depress glutamate-induced elevation in intracellular calcium concentration ([Ca²⁺]_c). The enzymatic assay indicated that COSs antagonized glutamate-evoked activation of caspase-3. These results collectively suggest that COSs prevent cultured hippocampal neurons from glutamate-induced cell damage by interfering with an increase in [Ca²⁺]_c and inhibiting caspase-3 activity.     

Neuropeptide Y protects retinal neural cells against cell death induced by ecstasy

Ecstasy (3,4-methylenedioxymethamphetamine; MDMA) has potent CNS stimulant effects. Besides the acute effects of MDMA, such as psychomotor activation, euphoria, decreased appetite, and hyperthermia, long-term damage of dopaminergic and serotonergic nerve terminals in multiple brain areas have also been reported. Although some studies have demonstrated that considerable amounts of MDMA reach the vitreous humor of the eye, and that serious visual consequences can result from MDMA consumption, the toxic effect of MDMA on the retina has not been completely elucidated. Neuropeptide Y (NPY) is present in the CNS, including the retina. The aim of the present study was to evaluate the effect of MDMA on rat retinal neural cell viability and investigate the involvement of 5-HT 2A-receptor (5-HT(2A)) activation. Moreover, the neuroprotective role of NPY on MDMA-induced toxicity was also investigated. MDMA induced necrosis [MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and propidium iodide assays] and apoptosis (immunoreactivity of cleaved caspase-3) in mixed cultures of retinal neural cells (neurons, macroglia and microglia), in a concentration-dependent manner. MDMA-induced toxicity was enhanced at higher temperature (40 degrees C) and was reduced by the 5HT(2A)-receptor antagonist, ketanserin (1 microM). Interestingly, necrotic and apoptotic cell death induced by MDMA was inhibited by NPY (100 nM).     

Normotonic cell shrinkage induced by Na+ deprivation results in apoptotic cell death in human epithelial HeLa cells

Apoptosis is a major form of cell death that occurs in response to a variety of signals in both physiological and pathological situations. A hallmark of apoptosis is normotonic cell shrinkage, called apoptotic volume decrease (AVD), the process of which involves fluxes of K(+), Cl(-), and Na(+). Na(+) influx was suggested to be required in Fas-induced apoptosis in human Jurkat T cells, whereas Na(+) efflux was found to be associated with AVD and apoptosis in human HL-60 cells. Here we examined the effects of extracellular Na(+) deprivation on cell volume and viability in human epithelial HeLa cells. The incubation of HeLa cells in normotonic Na(+)-free Ringer solution resulted in persistent cell shrinkage after > or = 30 min and reduction in cell viability after > or = 1 h. After exposure to Na(+)-free solution for 5 h, a marked reduction in cell viability was found to be associated with an activation of caspase-3 without showing significant LDH release, indicating that the cells underwent apoptosis but not necrosis. Na(+) deprivation-induced cell shrinkage and apoptotic cell death were significantly inhibited by a blocker of Na(+)-K(+)-2Cl(-) cotransporter (NKCC) or of the reverse-mode operation of Na(+)/Ca(2+) exchanger (NCX), but not by a blocker of Na(+)/H(+) exchanger (NHE). Therefore it is concluded that Na(+) deprivation causes persistent cell shrinkage resulting from Na(+) efflux mainly via NKCC and NCX and thereafter leads to apoptotic death of HeLa cells. It is also suggested that normotonic cell shrinkage per se, if persistent, provides a sufficient condition for apoptosis induction.     

Non-competitive antagonists of N-methyl-D-aspartate receptors protect cortical and hippocampal cell cultures against glutamate neurotoxicity

Brief exposure of cortical or hippocampal cell cultures to glutamate (500 microM) resulted in a progressive neuronal necrosis which is maximal 18-24 h later. Pretreatment of the cultures by a phencyclidine derivative: thienylphencyclidine (TCP), or the TCP precursor: GK86, or MK801 protected against glutamate toxicity. Non-competitive antagonists of N-methyl-D-aspartate receptors appear as potent tools for the in vitro protection of neuronal cells against excitatory amino acid toxicity.     

Evidence of apoptotic cell death in HIV encephalitis.

The mechanism of cell death in the brains of patients with acquired immune deficiency syndrome was examined in 15 cases, 8 of whom had human immunodeficiency virus (HIV) encephalitis, and in 8 control cases. Postmortem formalin-fixed, paraffin-embedded sections were prepared for routine histology and immunohistochemistry to detect cell-specific antigens. Apoptosis was detected by its morphology and by in situ end labeling of its characteristic oligonucleosomal fragments. Combined in situ end labeling and immunohistochemistry identified specific cell types. Six acquired immune deficiency syndrome brains, 5 of which had HIV encephalitis, contained positive nuclei by in situ end labeling. Co-labeling studies identified the cells as neurons, reactive astrocytes, and, rarely, the multinucleated giant cells of HIV encephalitis. The only control with nuclei positive by in situ end labeling had hepatic encephalopathy and Alzheimer type II astrocytes; the location and absence of cell-specific markers suggested a glial origin for the labeled cells. These results demonstrate that at least some neuronal and astrocytic death in HIV infection occurs by apoptosis. Its stimuli are unknown, but likely candidates include tumor necrosis factor or HIV viral products. Additionally, we hypothesize that apoptotic death of reactive astrocytes may be a normal mechanism whereby the brain removes an excess number of astrocytes that have proliferated after certain types of brain injury.     

Adenoviral inhibitors of apoptotic cell death

Adenoviruses (Ads) are endemic in the human population and the well-studied group C Ads typically cause an acute infection in the respiratory epithelium. A growing body of evidence suggests that these viruses also establish a persistent infection. The Ad genome encodes several proteins that counteract the host anti-viral mechanisms, which function to limit viral infections. This review describes the adenovirus immuno-regulatory proteins and how they function to block apoptosis of infected cells. In addition to facilitating the successful completion of the viral replication cycle and spread of progeny virus, these functions may help maintain the virus in a persistent state.     

Flow cytometry of apoptotic cell death

The term apoptosis or programmed cell death defines a genetically encoded cell death program, which is morphologically and biochemically distinct from necrosis or accidental cell death. The characteristic morphological signs of apoptosis (cellular shrinkage, membrane blebbing, nuclear condensation and fragmentation) are the final results of a complex biochemical cascade of events which is an integral part of physiological homeostasis. Techniques designed to identify, quantitate and characterize apoptosis are numerous, but flow cytometry (FCM) remains the methodology of choice to study the apoptotic cascade in relation to cell type, trigger and time. This review outlines the main stages of the apoptotic cascade together with current FCM methods. All FCM apoptosis assays described have a solid experimental basis and have been used successfully in basic research on molecular and biochemical mechanisms of apoptosis. In various clinical settings the ability to follow the apoptotic process in patient samples may offer the rationale for optimal treatment schedules.     

ROS-mediated apoptotic cell death in prostate cancer LNCaP cells induced by biosurfactant stabilized CdS quantum dots

Cadmium sulfide (CdS) quantum dots (QDs) have raised great attention because of their superior optical properties and wide utilization in biological and biomedical studies. However, little is known about the cell death mechanisms of CdS QDs in human cancer cells. This study was designed to investigate the possible mechanisms of apoptosis induced by biosurfactant stabilized CdS QDs (denoted as "bsCdS QDs") in human prostate cancer LNCaP cells. It was also noteworthy that apoptosis correlated with reactive oxygen species (ROS) production, mitochondrial damage, oxidative stress and chromatin condensation in a dose- and time-dependent manner. Results also showed involvement of caspases, Bcl-2 family proteins, heat shock protein 70, and a cell-cycle checkpoint protein p53 in apoptosis induction by bsCdS QDs in LNCaP cells. Moreover, pro-apoptotic protein Bax was upregulated and the anti-apoptotic proteins, survivin and NF-κB were downregulated in bsCdS QDs exposed cells. Protection of N-acetyl cysteine (NAC) against ROS clearly suggested the implication of ROS in hyper-activation of apoptosis and cell death. It is encouraging to conclude that biologically stabilized CdS QDs bear the potential of its applications in biomedicine, such as tumor therapy specifically by inducing caspase-dependent apoptotic cell death of human prostate cancer LNCaP cells.     

Protection against beta-amyloid induced abnormal synaptic function and cell death by Ginkgolide J

A new Ginkgo biloba extract P8A (TTL), 70% enriched with terpene trilactones, prevents A beta(1-42) induced inhibition of long-term potentiation in the CA1 region of mouse hippocampal slices. This neuroprotective effect is attributed in large part to ginkgolide J that completely replicates the effect of the extract. Ginkgolide J is also capable of inhibiting cell death of rodent hippocampal neurons caused by A beta(1-42). This beneficial and multi-faceted mode of action of the ginkgolide makes it a new and promising lead in designing therapies against Alzheimer's disease.     

A1 demonstrates restricted tissue distribution during embryonic development and functions to protect against cell death.

Members of the bcl-2 gene family are essential regulators of cell survival in a wide range of biological processes. A1, a member of the family, is known to be expressed in certain adult tissues. However, the precise tissue distribution and function of A1 remains poorly understood. We show here that A1 is expressed in multiple tissues during murine embryonic development. In the embryo, A1 was detected first at embryonic day 11.5 in liver, brain, and limbs. At day 13.5 of gestation, A1 expression was observed in the central nervous system, liver, perichondrium, and digital zones of developing limbs in a pattern different from that of bcl-X. In the central nervous system of 15.5-day embryos, A1 was expressed at high levels in the ventricular zone and cortical plate of brain cortex. Significantly, the interdigital zones of limbs and the intermediate region of the developing brain cortex, two sites associated with extensive cell death, were devoid of A1 and bcl-X. The expression of A1 was retained in many adult tissues. To assess the ability of A1 to modulate cell death, stable transfectants expressing different amounts of A1 protein were generated in K562 cells. Expression of A1 was associated with retardation of apoptotic cell death induced by actinomycin D and cycloheximide as well as by okadaic acid. Confocal microscopy showed that the A1 protein was localized to the cytoplasm in a pattern similar to that of Bcl-2. These results demonstrate that the expression of A1 is wider than previously reported in adult tissues. Furthermore, its distribution in multiple tissues of the embryo suggests that A1 plays a role in the regulation of physiological cell death during embryonic development.     

Immunosuppressant deoxyspergualin induces apoptotic cell death in dividing cells.

Deoxyspergualin (DSG) has been found to have an antitumour and immunosuppressive activity. However, the precise mechanism of action of DSG has not been clarified. We have used its analogue, methyldeoxyspergualin (MeDSG) for in vitro culture studies of DSG since it shows good stability in aqueous solution and retains strong immunosuppressive activity. In the present study, we found that MeDSG inhibited proliferation of rapidly dividing murine T-cell hybridomas, resulting in cell death. The cell death was accompanied by chromatin condensation and DNA cleavage at the linker regions between nucleosomes. Furthermore, MeDSG induced a reduction in mitochondrial transmembrane potential. When murine thymocytes were treated with MeDSG for 48 hr, a slight increase of DNA fragmentation was constantly observed, and selective depletion of CD4- CD8- cells was noticed. In contrast, CD4+ CD8+ cells were hardly affected. Moreover, splenic T-cells are resistant to MeDSG-induced apoptosis, as evaluated by measuring DNA cleavage. Our findings may account for the immunosuppressive and antitumour properties of DSG which were described in a number of previous studies.     

Inhibitory effect of the immunosuppressant FK506 on apoptotic cell death induced by HIV-1 gp120

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 may play a central role in inducing immunoregulatory disorders after HIV infection. The apoptotic death of normal human peripheral blood mononuclear cells was induced by priming with gp120 followed by stimulation with an anti-T cell receptor (TCR) antibody. Tumor necrosis factor- produced by gp120-binding macrophages may be important to induce this cell death. Treatment of gp120-primed cells with an immunosuppressant (FK506) before TCR signaling inhibited apoptotic cell death, and this blocking effect of FK506 was concentration dependent. FK506 did not have any influence on cell growth and viability over the range of concentrations tested. These findings suggest that FK506 is a potentially useful drug in delaying the onset of AIDS after HIV infection.     

Heat shock protein 60 from Klebsiella pneumoniae protects mononuclear cells from apoptotic cell death induced by dexamethasone

Aims

Bacterial heat shock proteins can have anti-apoptotic effects on human cells. We investigated whether enterobacterial HSP60 can protect peripheral blood mononuclear cells (PBMC) from DXM-induced apoptosis and if this effect requires cytoskeleton participation.

Main methods

Anti-apoptotic effect from enterobacterial HSP60 was analyzed by adding these proteins to peripheral mononuclear cells cultures before DXM induction. Percentage of apoptotic cells was determined by SubG0 peak and TUNEL techniques in a flow cytometer.

Key findings

Our results showed significant protective effect of HSP60 Klebsiella pneumoniae and E. coli, in the DXM-induced apoptosis in PBMC. Similar results were obtained with recombinant human HSP60. The same protective effect of proteins was observed in CD4+ and CD8 + T cell subpopulations. To analyze if enterobacterial HSP60 need internalization to have the anti-apoptotic effect, we used cytoskeleton inhibitors such as: nocodazole, cytochalasin D and amiloride, the three inhibitors significantly affected the protective role of HSP60 in apoptosis induced with DXM. Results suggest that the protective effect of HSP60 K. pneumoniae and E. coli requires the participation of contractile structures for the internalization of this protein by the cells, we suggest that the internalization of enterobacterial HSP60 could be carry out by macropinocytosis.

Significance

We report for the first time that K. pneumoniae and E. coli HSP60 have protective effect in the apoptosis induced with DXM in PBMC from healthy subjects and that this effect requires the internalization of the protein with active participation of the cytoskeleton.     

Hepatitis C virus NS5A protein protects against TNF-alpha mediated apoptotic cell death

Hepatitis C virus (HCV) often causes a prolonged and persistent infection which may lead to hepatocellular carcinoma. We have previously reported that the nonstructural 5A (NS5A) protein of HCV promotes cell growth [Ghosh, A.K., Steele, R., Meyer, K., Ray, R., Ray, R.B., 1999. Hepatitis C virus NS5A protein modulates cell cycle regulatory genes and promotes cell growth. J. Gen. Virol. 80, 1179-1183]. In this study, we investigated the role of HCV NS5A (genotype 1a, strain H) in TNF-alpha induced apoptotic cell death. HepG2 cells expressing NS5A exhibited an inhibitory role in relation to TNF-alpha mediated apoptotic cell death. The NS5A protein blocked the activation of caspase-3 and inhibited proteolytic cleavage of the death substrate poly (ADP-ribose) polymerase in TNF-alpha induced cells. Together, these results suggest that HCV NS5A protein protects against TNF-alpha mediated apoptotic cell death.     

Alginate oligosaccharide protects against endoplasmic reticulum- and mitochondrial-mediated apoptotic cell death and oxidative stress

Oxidative stress is a major component of harmful cascades activated in neurodegenerative disorders. We sought to elucidate possible effects of alginate oligosaccharide (AOS) on H(2)O(2)-induced cell death and to determine the underlying molecular mechanisms in neuron-like PC12 cells. We found that AOS treatment protected PC12 cells against H(2)O(2)-induced endoplasmic reticulum (ER) and mitochondrial-dependent apoptotic cell death. AOS promoted Bcl-2 expression, while blocked Bax expression and inhibited H(2)O(2)-induced caspase-3 activation. It also blocked PARP cleavage. AOS acted on key molecules in apoptotic cell death pathway and reduced p53, p38, c-June NH2-terminal kinase phosphorylations, inhibited NFkB, and enhanced Nrf2 activation. These results suggest that treatment of PC12 cells with AOS can block H(2)O(2)-induced oxidative stress and caspase-dependent apoptotic cascades originating from both ER and mitochondria. Our in?vivo experiments further confirm the neuroprotective potential of AOS against Aβ-induced neural damage. According to our data, the involvement of caspase-independent pathway in AOS-induced protection appears to be unlikely.     

Mechanism of lymphoma cell death induced by cyclic AMP.

A mouse lymphoma tissue culture line, S49, is killed by isoproterenol, choleratoxin, or prostaglandin E1, inducers of cyclic AMP (cAMP) in these cells, or by the analog dibutyryl (db) cAMP. Cell death follows arrest in the G1 phase of the cell cycle. Mutant subclones obtained by growing S49 with dbcAMP were resistant to killing. They were deficient in cAMP-dependent protein kinase. These results are discussed in relation to the possible physiologic role of cAMP-induced cell death in T-cell differentiation.