Involvement of miltefosine-mediated ERK activation in glioma cell apoptosis through Fas regulation

The anti-neoplastic property of alkyl phospholipids has been tested for the treatment of several malignancies. In this study, we evaluated the efficacy of miltefosine (Hexadecylphosphocholine--an alkyl phospholipids analogue) on glioblastoma multiforme. In this study, we demonstrate that miltefosine-induced apoptosis is accompanied by elevated Fas, Fas-associated death domain (FADD) expression, caspase-8 activity and the increased distribution of Fas and FADD towards lipid raft microdomain to form death inducing signaling complex. Treatment with miltefosine resulted in increase in Ras, extracellular signal-regulated kinase (ERK) and p38MAPK activity. Expression of dominant-negative Ras (Ras N17) attenuated miltefosine-mediated apoptosis. Although inhibition of both ERK and p38MAPK decreased the pro-apoptotic effects of miltefosine, it was the inhibition of ERK and not p38MAPK activation that decreased Fas and FADD expression. An ERK-dependent increase in the expression of gammaH2AX-involved in response to DNA double-stranded breaks was also observed. Taken together, our findings suggest the involvement of ERK activation in miltefosine-induced glioma cell apoptosis.     

Brij detergents reveal new aspects of membrane microdomain in erythrocytes

Membrane microdomains enriched in cholesterol, sphingolipids (rafts), and specific proteins are involved in important physiological functions. However their structure, size and stability are still controversial. Given that detergent-resistant membranes (DRMs) are in the liquid-ordered state and are rich in raft-like components, they might correspond to rafts at least to some extent. Here we monitor the lateral order of biological membranes by characterizing DRMs from erythrocytes obtained with Brij-98, Brij-58, and TX-100 at 4?°C and 37?°C. All DRMs were enriched in cholesterol and contained the raft markers flotillin-2 and stomatin. However, sphingomyelin (SM) was only found to be enriched in TX-100-DRMs – a detergent that preferentially solubilizes the membrane inner leaflet – while Band 3 was present solely in Brij-DRMs. Electron paramagnetic resonance spectra showed that the acyl chain packing of Brij-DRMs was lower than TX-100-DRMs, providing evidence of their diverse lipid composition. Fatty acid analysis revealed that the SM fraction of the DRMs was enriched in lignoceric acid, which should specifically contribute to the resistance of SM to detergents. These results indicate that lipids from the outer leaflet, particularly SM, are essential for the formation of the liquid-ordered phase of DRMs. At last, the differential solubilization process induced by Brij-98 and TX-100 was monitored using giant unilamellar vesicles. This study suggests that Brij and TX-100-DRMs reflect different degrees of lateral order of the membrane microdomains. Additionally, Brij DRMs are composed by both inner and outer leaflet components, making them more physiologically relevant than TX-100-DRMs to the studies of membrane rafts.     

The critical role of protein kinase C-theta in Fas/Fas ligand-mediated apoptosis

A functional immune system not only requires rapid expansion of antigenic specific T cells, but also requires efficient deletion of clonally expanded T cells to avoid accumulation of T cells. Fas/Fas ligand (FasL)-mediated apoptosis plays a critical role in the deletion of activated peripheral T cells, which is clearly demonstrated by superantigen-induced expansion and subsequent deletion of T cells. In this study, we show that in the absence of protein kinase C-theta (PKC-theta), superantigen (staphylococcal enterotoxin B)-induced deletion of Vbeta8(+) CD4(+) T cells was defective in PKC-theta(-/-) mice. In response to staphylococcal enterotoxin B challenge, up-regulation of FasL, but not Fas, was significantly reduced in PKC-theta(-/-) mice. PKC-theta is thus required for maximum up-regulation of FasL in vivo. We further show that stimulation of FasL expression depends on PKC-theta-mediated activation of NF-AT pathway. In addition, PKC-theta(-/-) T cells displayed resistance to Fas-mediated apoptosis as well as activation-induced cell death (AICD). In the absence of PKC-theta, Fas-induced activation of apoptotic molecules such as caspase-8, caspase-3, and Bid was not efficient. However, AICD as well as Fas-mediated apoptosis of PKC-theta(-/-) T cells were restored in the presence of high concentration of IL-2, a critical factor required for potentiating T cells for AICD. PKC-theta is thus required for promoting FasL expression and for potentiating Fas-mediated apoptosis.     

Estrogen‐regulated developmental neuronal apoptosis is determined by estrogen receptor subtype and the Fas/Fas ligand system

Adult sexual dimorphism in neuronal cell number is controlled by estrogen exposure during a tightly defined period of rat brain development. The mechanisms of estrogen's effect are unknown; one possibility is regulation of programmed cell death (apoptosis). In this study we have shown that estradiol can function as a neuroprotective agent or an inducer of apoptosis, depending on the estrogen receptor‐subtype present in the cell. Thus, ERα has a neuroprotective effect, while ERβ mediates the induction of apoptosis in neuronal cells. Moreover, we show that estrogen‐induced apoptosis through ER‐β requires the expression of Fas‐ and Fas ligand (FasL) proteins, since the absence of FasL in neurons prevents this effect. Furthermore, we demonstrate that microglia‐secreted products induce the expression of FasL necessary to mediate estradiol–ERβ apoptotic effect. These findings may explain the dichotomous effect of fetal estradiol on the adult neuronal number. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 64–78, 2000     

Amyloid beta-peptide 31-35-induced neuronal apoptosis is mediated by caspase-dependent pathways via cAMP-dependent protein kinase A activation

This study aims to investigate the roles of the protein kinase A (PKA)- and caspase-dependent pathways in amyloid beta-peptide 31-35 (Abeta[31-35])-induced apoptosis, and the mechanisms of neuroprotection by group III metabotropic glutamate receptor (mGluR) activation against apoptosis induced by Abeta[31-35] in cortical neurons. We demonstrated that Abeta[31-35] induces neuronal apoptosis as well as a significant increase in caspase-3, -8 and -9. Activation of group III mGluRs by l-serine-O-phosphate and (R,S)-4-phosphonophenylglycine (two group III mGluR agonists), which attenuate the effects of Abeta[31-35], provides neuroprotection to the cortical neurons subjected to Abeta[31-35]. We also showed that Rp-cAMP, an inhibitor of cAMP-dependent PKA, has the ability to protect neurons from Abeta[31-35]-induced apoptosis and to reverse almost completely the effects of Abeta[31-35] on the activities of caspase-3. Further, we found that Sp-cAMP, an activator of cAMP-dependent PKA, can significantly abolish the l-serine-O-phosphate- and (R,S)-4-phosphonophenylglycine-induced neuroprotection against apoptosis, and decrease caspase-3, -8 and -9 in the Abeta[31-35]-treated neurons. Our findings suggest that neuronal apoptosis induced by Abeta[31-35] is mediated by the PKA-dependent pathway as well as the caspase-dependent intrinsic and extrinsic apoptotic pathways. Activation of group III mGluRs protects neurons from Abeta[31-35]-induced apoptosis by blocking the caspase-dependent pathways. Inhibition of the PKA-dependent pathway might also protect neurons from Abeta[31-35]-induced apoptosis by blocking the caspase-dependent pathways. Taken together, our observations suggest that Abeta[31-35] might have the ability to activate PKA, which in turn activates the caspase-dependent intrinsic and extrinsic apoptotic pathways, inducing apoptosis in the cortical neurons.     

Soluble Fas (FasB) regulates luteal cell apoptosis during luteolysis in murine ovaries

During luteolysis, luteal cell apoptosis is induced by the Fas ligand (FasL)/Fas system. In murine luteal bodies, we demonstrated the expression of mRNA of soluble form of Fas (FasB), which binds to FasL and prevents apoptosis induction. By in situ hybridization, strong expression of FasB mRNA was observed in normal luteal bodies, in which no apoptotic cells were detected, but negative/trace expression in regressing luteal bodies, in which many apoptotic cells were observed. Immunohistochemical staining revealed that Fas and TNF-alpha were localized in both normal and regressing luteal bodies, but IFN-gamma was localized only in regressing luteal bodies. Apoptosis was induced in primary cultured luteal cells, when they were pretreated with TNF-alpha and IFN-gamma and then incubated with TNF-alpha, IFN-gamma, and mouse recombinant FasL (rFasL). However, no apoptosis was detected in the cells, when they were treated with rFasL alone, TNF-alpha alone, IFN-gamma alone, TNF-alpha and rFasL, IFN-gamma and rFasL, or TNF-alpha and IFN-gamma. Fas mRNA expression in cultured luteal cells was up-regulated by the treatment of TNF-alpha, IFN-gamma, or TNF-alpha and IFN-gamma. The expression of FasB mRNA was down-regulated, when the cells were treated with TNF-alpha and IFN-gamma, but its expression was not changed by the treatment of TNF-alpha alone or IFN-gamma alone. We conclude that FasB inhibits the apoptosis induction in luteal cells of normal luteal bodies, and that decreased FasB production induced by TNF-alpha and IFN-gamma made possible the apoptosis induction in the luteal cells of regressing luteal bodies.     

Reorganization of prion protein membrane environment during low potassium-induced apoptosis in primary rat cerebellar neurons

We studied the changes occurring in the membrane environment of prion protein (PrP) during apoptosis induced by low potassium in primary rat cerebellar neurons. Ceramide levels increased during apoptosis-inducing treatment, being doubled with respect to time-matched controls after 24 h. Sphingomyelin levels were parallely decreased, while cholesterol and ganglioside contents were not affected. Changes in ceramide and sphingomyelin composition were exclusively restricted to a detergent-resistant membrane fraction. The pro-apoptotic treatment was accompanied by the down-regulation of PrP and of the non-receptor kinase Fyn. The levels of PrP and Fyn were correspondingly reduced in the detergent-resistant membrane fraction. In control cells, the membrane microenvironment separated by immunoprecipitation with anti-PrP antibody contained 80% of the detergent-resistant PrP and 35% and 38% of the sphingolipids and cholesterol respectively. Upon low potassium treatment, 20% of the PrP originally present in the detergent-resistant fraction was immunoprecipitated, together with 19% of sphingolipids and 22% of cholesterol. Thus, PrP in the immunoprecipitate from apoptotic cells was ninefold less than in control ones, while sphingolipids and cholesterol were about 50% with respect to controls cells. The molar ratio between cholesterol, sphingomyelin and ceramide was 15 : 6 : 1 in the PrP-rich environment from control neurons, and 6 : 2 : 1 in that from apoptotic cells.     

Double role of Fas ligand in the apoptosis of intervertebral disc cells in vitro

Fas ligand （FasL） may play an important role in maintaining the immune privilege of intervertebral disc （IVD）. Besides, it is closely related to the apoptosis of degenerative disc cells. Nowadays, lots of reports have described about the paradoxical effects of FasL, although the effect of FasL on IVD cells is still under debate. In this study, we tried to investigate the effects of FasL on Fas expression and on the apoptosis of nucleus pulposus （NP） cells in Sprague-Dawley rats. The results showed that the expression of Fas in NP cells was significantly increased by the recombinant FasL. Meanwhile, the apoptosis of NP cells increased markedly in a FasL dose-dependent manner. Interestingly, RNA interference results indicated that the increase of Fas expression and the NP cell apoptosis described previously were inhibited by Fas siRNA, suggesting that RNA interference might be one of novel strategies to prevent IVD cells from apoptosis.     

Activated protein C via PAR1 receptor regulates survival of neurons under conditions of glutamate excitotoxicity

The effect of an anticoagulant and cytoprotector blood serine proteinase--activated protein C (APC)--on survival of cultured hippocampal and cortical neurons under conditions of glutamate-induced excitotoxicity has been studied. Low concentrations of APC (0.01-10 nM) did not cause neuron death, but in the narrow range of low concentrations APC twofold and stronger decreased cell death caused by glutamate toxicity. High concentrations of APC (>50 nM) induced the death of hippocampal neurons similarly to the toxic action of glutamate. The neuroprotective effect of APC on the neurons was mediated by type 1 proteinase-activated receptor (PAR1), because the inactivation of the enzyme with phenylmethylsulfonyl fluoride or PAR1 blockade by a PAR1 peptide antagonist ((Tyr1)-TRAP-7) prevented the protective effect of APC. Moreover, APC inhibited the proapoptotic effect of 10 nM thrombin on the neurons. Geldanamycin, a specific inhibitor of heat shock protein Hsp90, completely abolished the antiapoptotic effect of 0.1 nM APC on glutamate-induced cytotoxicity in the hippocampal neurons. Thus, APC at low concentrations, activating PAR1, prevents the death of hippocampal and cortical neurons under conditions of glutamate excitotoxicity.     

Human DU145 prostate cancer cells overexpressing mitogen-activated protein kinase phosphatase-1 are resistant to Fas ligand-induced mitochondrial perturbations and cellular apoptosis

Recent studies have suggested that MAP kinase phosphatase 1 (MKP-1) is overexpressed in prostate cancer. To evaluate the role of MKP-1 in regulating cell death and tumor growth in prostate cancer, MKP-1 was conditionally overexpressed in the human prostate cancer cell line DU145. Overexpression of MKP-1 in DU145 cells blocked activation of stress-activated protein kinase (SAPK/JNK). MKP-1 overexpression in DU-145 cells was also found to inhibit Fas ligand (FasL)-induced apoptosis, as well as block the activation of caspases by Fas engagement. In addition, MKP-1 blocked the activation of apoptosis by transfected MEKK-1 and ASK-1, presumably through its inhibition of the SAPK/JNK family of enzymes. MKP-1 blocked the ability of FasL to induce loss of mitochondrial transmembrane potential (_m), suggesting that MKP-1 acts upstream of mitochondrial pro-apoptotic events induced by FasL and that the SAPK/JNK pathway may form the signaling link between Fas receptor and mitochondrial dysfunction. Thus, MKP-1 overexpression in prostate cancer may play a role in promoting prostate carcinogenesis by inhibiting FasL-induced cell death.     

Asymmetric dimethylarginine attenuates serum starvation-induced apoptosis via suppression of the Fas (APO-1/CD95)/JNK (SAPK) pathway

Asymmetric dimethylarginine (ADMA) is synthesized by protein arginine methyltransferases during methylation of protein arginine residues and released into blood upon proteolysis. Higher concentrations of ADMA in blood have been observed in patients with metabolic diseases and certain cancers. However, the role of ADMA in colon cancer has not been well investigated. ADMA serum levels in human patients diagnosed with colon cancer were found to be higher than those present in healthy subjects. ADMA treatment of LoVo cells, a human colon adenocarcinoma cell line, attenuated serum starvation-induced apoptosis and suppressed the activation of the Fas (APO-1/CD95)/JNK (SAPK) (c-Jun N terminal protein kinase/stress-activated protein kinase)pathway. ADMA also suppressed the activation of JNK triggered by death receptor ligand anti-Fas mAb and exogenous C₂-ceramide. Moreover, we demonstrated that ADMA pretreatment protected LoVo cells from doxorubicin hydrochloride-induced cell death and activation of the Fas/JNK pathway. In summary, our results suggest that the elevated ADMA in colon cancer patients may contribute to the blocking of apoptosis of cancer cells in response to stress and chemotherapy.     

Normal breast epithelial cells induce apoptosis of breast cancer cells via Fas signaling

Fas/Fas ligand (Fas L) death pathway is an important mediator of apoptosis. Deregulation of Fas pathway is reported to be involved in the immune escape of breast cancer and the resistance to anti-cancer drugs. In this study, we demonstrated that conditioned medium by normal breast epithelial cells (NBEC-CM) induced apoptosis of MCF-7 and T-47D Fas-sensitive cells but had no effect on MDA-MB-231 Fas-resistant cells. Inhibition of PI3 kinase or NF-kappaB by specific inhibitors or transient transfections restored the sensitivity of MDA-MB-231 cells to NBEC-induced apoptosis. Moreover, the constitutive activation of NF-kappaB was controlled by PI3 kinase because inhibition of PI3 kinase reduced NF-kappaB activity. Inducible activation of NF-kappaB rendered MCF-7 cells resistant to NBEC-CM- and Fas agonist antibody-triggered apoptosis. Therefore, constitutive or inducible activation of PI3 kinase and/or NF-kappaB in breast cancer cells rendered them resistant to NBEC-triggered apoptosis. In addition, Fas neutralizing antibody and dominant negative Fas abolished NBEC-triggered apoptosis. Western blot and confocal microscopy analysis showed an increase of membrane Fas/Fas L when cells were induced into apoptotis by NBEC-CM. Taken together, these data show that NBEC induced apoptosis in breast cancer cells via Fas signaling.     

Pink1 protects cortical neurons from thapsigargin-induced oxidative stress and neuronal apoptosis

Apoptosis mediates the precise and programmed natural death of neurons and is a physiologically important process in neurogenesis during maturation of the central nervous system. However, premature apoptosis and/or an aberration in apoptosis regulation are implicated in the pathogenesis of neurodegeneration. Thus, it is important to identify neuronal pathways/factors controlling apoptosis. Pink1 [phosphatase and tensin homologue (PTEN)-induced kinase 1] is a ubiquitously expressed gene and has been reported to have a physiological role in mitochondrial maintenance, suppressing mitochondrial oxidative stress, fission and autophagy. However, how Pink1 is involved in neuronal survival against oxidative stress remains not well understood. In the present paper, we demonstrate that thapsigargin, a specific irreversible inhibitor of endoplasmic reticulum (ER) calcium-ATPase, could lead to dramatic oxidative stress and neuronal apoptosis by ectopic calcium entry. Importantly, the neuronal toxicity of thapsigargin inhibits antioxidant gene Pink1 expression. Although Pink1 knockdown enhances the neuronal apoptosis by thapsigargin, its overexpression restores it. Our findings have established the neuronal protective role of Pink1 against oxidative stress and afford rationale for developing new strategy to the therapy of neurodegenerative diseases.     

Role of apoptosis and Fas/FasL system in the oogenesis of the spotted ray Torpedo marmorata

In the present paper we investigated the role played by apoptosis during oogenesis in the cartilaginous fish Torpedo marmorata. TEM, TUNEL and immunohistochemical techniques were employed to specifically reveal morphological and biochemical hallmarks of apoptosis in specimens from birth to sexual maturity. Data obtained demonstrate that apoptosis occurs in prefollicular oocyte selection, in maintaining the homeostasis of granulosa in healthy growing oocyte and in resorbing atretic follicles. In this respect, the involvement of apoptosis in Torpedo marmorata oogenesis closely parallels that found in mammals, thus confirming that strategies of germ cell selection among vertebrates have been evolutionarily preserved.