Activation Of Apoptotic Cascade Research Articles

Nicotinamide inhibits alkylating agent-induced apoptotic neurodegeneration in the developing rat brain.

BackgroundExposure to the chemotherapeutic alkylating agent thiotepa during brain development leads to neurological complications arising from neurodegeneration and irreversible damage to the developing central nerve system (CNS). Administration of single dose of thiotepa in 7-d postnatal (P7) rat triggers activation of apoptotic cascade and widespread neuronal death. The present study was aimed to elucidate whether nicotinamide may prevent thiotepa-induced neurodegeneration in the developing rat brain.Methodology/Principal FindingsNeuronal cell death induced by thiotepa was associated with the induction of Bax, release of cytochrome-c from mitochondria into the cytosol, activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP-1). Post-treatment of developing rats with nicotinamide suppressed thiotepa-induced upregulation of Bax, reduced cytochrome-c release into the cytosol and reduced expression of activated caspase-3 and cleavage of PARP-1. Cresyl violet staining showed numerous dead cells in the cortex hippocampus and thalamus; post-treatment with nicotinamide reduced the number of dead cells in these brain regions. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) and immunohistochemical analysis of caspase-3 show that thiotepa-induced cell death is apoptotic and that it is inhibited by nicotinamide treatment.ConclusionNicotinamide (Nic) treatment with thiotepa significantly improved neuronal survival and alleviated neuronal cell death in the developing rat. These data demonstrate that nicotinamide shows promise as a therapeutic and neuroprotective agent for the treatment of neurodegenerative disorders in newborns and infants.

Abstract 5480: Effect of the Smac mimetic TL32711 in combination with TRAIL or TNF alpha on a panel of melanoma cell lines

Abstract Although major advances have been made recently in the treatment of malignant melanoma, it remains an incurable disease when not surgically excised at a very early stage. This is due to the development of resistance to targeted compounds currently being tested in patients. In addition, the genetic heterogeneity of melanoma often limits the effects of those therapies to specific subsets of patients. New targets that are exploitable in multiple genetic subgroups of melanoma and that can overcome resistance are therefore still urgently needed. IAP's (inhibitor of apoptosis proteins) have been described as major players in conferring resistance to therapy by blocking the apoptotic cascade in a high percentage of melanomas. Thus, the sensitivity to apoptotic stimuli, such as TNF alpha or TRAIL, could be restored in melanomas using the novel Smac (second mitochondria-derived activator of caspases) mimetic, TL32711. A panel of patient-derived human melanoma cell lines was clustered according to mutational status, representing all major genetic subgroups of cutaneous melanoma. Cells were treated with TL32711 as a single agent or in combination with TRAIL or TNF-α for 72h. Cell viability was assessed by MTS assay. Target inhibition and initiation of apoptosis were also evaluated. To more accurately predict in vivo efficacy, cells were grown as three dimensional spheroids in a collagen matrix and treated as described above. Treatment effects on spheroids were assessed through confocal microscopy using live/ dead immune fluorescent staining, as well as alamar blue cell viability assay for objective quantification.We observed that seventeen out of eighteen cell lines tested were resistant to TL32711 as a single agent in vitro, even at high doses of the compound. Similarly, treatment with TRAIL or TNF-α alone did not show any significant increase in apoptosis. However, when TL32711 was combined with either TRAIL or TNF-α, a strong synergistic activity was observed in twelve out of eighteen cell lines at low doses, resulting in a dramatic increase in cell death of adherent and three dimensional cultures (spheroids). In all treated cell lines, degradation of the target IAP proteins was observed. However, only in sensitive cell lines, activation of apoptotic cascade was observed.In conclusion, the Smac mimetic TL32711 exhibits strong synergistic activity in combination with TRAIL or TNF-α leading to apoptosis in the majority of the genetically diverse cell lines tested. Since TNF-α and TRAIL are present in a high percentage of melanoma tumors in vivo, single agent activity of TL32711 may be observed in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5480. doi:10.1158/1538-7445.AM2011-5480

Novel histone deacetylase inhibitor exhibits antitumor activity via apoptosis induction in oral squamous cell carcinoma

Epigenetic modifications such as histone deacetylation are commonly related to tumor development and histone deacetylase (HDAC) inhibitors have been shown to be potential drugs for cancer treatment. In the present study, we investigated the effects of a novel HDAC inhibitor, Ky-2, on oral squamous carcinoma cells in vitro. Cell viability was significantly reduced by treatment with Ky-2 at 25 nM, while it also led to augmentation of the proportion of cells in the sub-G1 phase and DNA fragmentation. In addition, immunoblot analysis revealed that Ky-2 enhanced the expression of apoptosis-related proteins. Our results showed that a low concentration of Ky-2 induced apoptosis in oral squamous carcinoma cells via activation of apoptotic cascades.

Selective Inhibition of HDAC6 with a New Prototype Inhibitor (ACY-1215) Overcomes Bortezomib Resistance In Multiple Myeloma (MM)

AbstractAbstract 2997HDAC enzymes, whose activity has been linked to the transcription of DNA in several cancers including multiple myeloma (MM), are being studied as novel therapeutic targets. Four classes of HDAC enzymes have been identified and several non-specific pan-HDAC and class I HDAC inhibitors have been evaluated in clinical studies. HDAC6, a class II HDAC, has recently been linked to the activity of aggresomes that degrade unfolded and misfolded ubiquitinated proteins. Importantly, targeting both proteasomal and aggresomal protein degradation systems with proteasome inhibitors and HDAC inhibitors respectively, induces accumulation of polyubiquitinated proteins, followed by activation of apoptotic cascades and synergistic cytotoxocity.Here we investigated the preclinical activity of an HDAC6 selective inhibitor ACY-1215 in MM, either alone or in combination with bortezomib. In vitro enzyme assays showed that ACY-1215 has potent inhibitory activity against HDAC6 (IC50 0.0054 μ M) compared to the other HDACs, including Class I HDACs. Maximal cytotoxicity of ACY-1215 against MM cell lines was observed at 48h, with IC50 values ranging from 2–8 μ M. ACY-1215 was also effective against patient MM cells, including bortezomib resistant MM cells, without significant cytotoxicity in normal peripheral blood mononuclear cells (PBMCs). Moreover, ACY-1215 in a dose-dependent manner inhibited DNA synthesis of MM cells at 48h triggered either by binding to bone marrow stromal cells (BMSCs) or by exogenous IL6 and IGF-1, confirming its ability to overcome the proliferative advantage conferred by BMSCs and cytokines. We next studied whether the highly selective HDAC6 inhibitor ACY-1215 could achieve the same efficacy as a pan-HDAC inhibitor such as SAHA, but with less toxicity. Compared to SAHA, ACY-1215 was not toxic to PHA stimulated PBMCs. Importantly, both ACY-1215 and SAHA induced dose-dependent acetylation of α-tubulin in MM.1S cells, but ACY-1215 induced less potent acetylation of lysine on histone H3 and histone H4 than SAHA, further confirming its specific inhibitory effect on HDAC6 activity.We next combined low doses of ACY-1215 to inhibit aggressomal with bortezomib to inhibit proteasomal protein degradation, and showed synergistic anti MM activity (Combination Index < 0.9), resulting in apoptosis via the activation of caspase-8,-9,-3 and poly (ADP) ribosome polymerase. Annexin V+PI+ staining after 24h treatment confirmed increased cells in early and late apoptosis after combined therapy (83.6%) compared to ACY-1215 1μ M (10.2%) or bortezomib 5 nM (36.6%) treatment alone. Since NF-kB pathway plays a crucial role in MM cell survival and bortezomib triggers NF-κB activity in some MM cell lines, we next investigated the effect of combined therapy on NF-κB activity. MM.1S cells were co-cultured with BMSCs and then treated with bortezomib 5nM, ACY-1215 1μ M, or the combination. Interestingly, bortezomib treatment significantly enhanced NF-κB activity in MM.1S cells co-cultured with BMSCs, whereas ACY-1215 treatment resulted in only a modest increase in NF-κB activity. Importantly, combined therapy was associated with partial abrogation of bortezomib-induced NF-κb activity. Ongoing studies are determining whether this effect on NF-κB activity contributes to the cytotoxic effect of ACY-1215/bortezomib combination therapy.Finally, we confirmed the synergistic anti-MM activity of ACY-1215 and bortezomib in vivo using two different xenograft mouse models: human MM injected subcutaneously (plasmacytoma model); and luciferase-expressing human MM injected intravenously (disseminated MM model). In the, plasmacytoma model, daily (5×/week) dosing of ACY-1215 (IP: 50 mg/kg) and bortezomib (IV: 0.5 mg/kg) given twice weekly significantly inhibited tumor growth (p<0.0001) and prolonged survival (34 days in the combined treatment group versus 22 days in the control group, p<0.0011). In the disseminated MM model, daily (5x/week) dosing of ACY-1215 (IP: 75 mg/kg) and bortezomib (IP: 1.5mg/kg) once weekly significantly inhibited tumor growth (p<0.0001) and prolonged survival (40 days in the combined treatment group versus 17 days in the control group, p<0.0001). Importantly, no significant adverse effects were noted in either in vivo model. These results therefore provide the preclinical rationale for clinical trials of ACY-1215 together with bortezomib in the treatment of MM. Disclosures:Jarpe:Acetylon: Employment. Anderson:MILLENNIUM: Consultancy; CELGENE: Consultancy; NOVARTIS: Consultancy; ONYX: Consultancy; MERCK: Consultancy; BMS: Consultancy; Acetylon: Membership on an entity's Board of Directors or advisory committees. Jones:Acetylon: Employment. Raje:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Astra Zeneca: Research Funding; Acetylon: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees.

A propionyloxy derivative of 11-keto-β-boswellic acid induces apoptosis in HL-60 cells mediated through topoisomerase I & II inhibition

Boswellic acids have invariably been reported for their antiproliferative potential in various cell systems. In the present study the growth inhibitory effect of propionyloxy derivative of 11-keto-β-boswellic acid (PKBA; a semisynthetic analogue of 11-keto-β-boswellic acid) on HL-60 promyelocytic leukemia cells is being reported for the first time. In the preliminary studies, in vitro cytotoxicity of PKBA was investigated against eight human cancer cell lines viz., IMR-32, SF-295 (both neuroblastoma), PC-3 (prostate), Colo-205 (colon), MCF-7 (breast), OVCAR-5 (ovary), HL-60, Molt-4 (both leukemia) and their respective IC50 values were found to be 5.95, 7.11, 15.2, 14.5, 15, 15.9, 8.7 & 9.5μg/ml, respectively. For determining the mechanism of cell death in HL-60 cells, PKBA was subjected to different mechanistic studies. DNA relaxation assay of PKBA revealed inhibition of both topoisomerases I & II. The fragmentation analysis of DNA revealed typical ladders indicating the cytotoxic effect to be mediated by induction of apoptosis. The morphologic studies of PKBA showed the presence of true apoptotic bodies. Apoptosis was confirmed further by flow-cytometric detection of sub-G1 peaks and enhanced annexin-V-FITC binding of the cells. The activation of apoptotic cascade by PKBA in HL-60 cells was found to be associated with the loss of mitochondrial membrane potential, release of cytochrome c, activation of initiator and executioner caspases and cleavage of poly ADP ribose polymerase (PARP). In vivo studies of PKBA revealed anti-tumoral activity against both ascitic and solid murine tumor models. These studies thus demonstrate PKBA to induce apoptosis in HL-60 cells due to the inhibition of topoisomerases I and II.

Protective effects of caspase-9 and poly(ADP-ribose) polymerase inhibitors on ischemia-reperfusion-induced myocardial injury

The present study was designed to investigate the cardio-protective effect of Ac-LEDH-cmk a selective caspase-9 inhibitor and 5-aminoisoquinolinone a selective Poly (ADP-ribose) polymerase inhibitor on ischemia and reperfusion induced apoptotic and necrotic cell death in rats. Isolated rat hearts were exposed to 30 minutes of global ischemia followed by 120 minutes of reperfusion using Langendorff's apparatus. Myocardial injury was assessed in the terms of infarct size, release of lactate dehydrogenase, creatine kinase enzymes and apoptotic index was assessed by DNA smearing on agarose gel electrophoresis. Pretreatments with specific inhibitor of caspase-9, Ac-LEHD-cmk (0.07 muM and 0.105 muM), and inhibitor of PARP, 5-aminoisoquinolinone (5 microM and 7.5 muM), significantly attenuated I/R induced increase in infarct size, release of lactate dehydrogenase and creatine kinase in the coronary effluent, and apoptotic index. Therefore, it may be concluded that inhibition of caspase-9 and PARP prevent ischemia and reperfusion-induced activation of apoptotic cascade and necrosis in rat myocardium.

Deleterious Role of TLR3 during Hyperoxia-induced Acute Lung Injury

Acute respiratory distress syndrome (ARDS) manifests clinically as a consequence of septic and/or traumatic injury in the lung. Oxygen therapy remains a major therapeutic intervention in ARDS, but this can contribute further to lung damage. Patients with ARDS are highly susceptible to viral infection and it may be due to altered Toll-like receptor (TLR) expression. To evaluate the role of TLR3 in ARDS. TLR3 expression and signaling was determined in airway epithelial cells after in vitro hyperoxia challenge. Using a murine model of hyperoxia-induced lung injury, the role of TLR3 was determined using either TLR3-gene deficient mice or a specific neutralizing antibody directed to TLR3. Increased TLR3 expression was observed in airway epithelial cells from patients with ARDS. Further, hyperoxic conditions alone were a major stimulus for increased TLR3 expression and activation in cultured human epithelial cells. Interestingly, TLR3(-/-) mice exhibited less acute lung injury, activation of apoptotic cascades, and extracellular matrix deposition after 5 days of 80% oxygen compared with wild-type (TLR3(+/+)) mice under the same conditions. Administration of a monoclonal anti-TLR3 antibody to TLR3(+/+) mice exposed to hyperoxic conditions likewise protected these mice from lung injury and inflammation. The potential for redundancy in function as well as cross-talk between distinct TLRs may indeed contribute to whether the inflammatory cascade can be effectively disrupted once signaling has been initiated. Together, these data show that TLR3 has a major role in the development of ARDS-like pathology in the absence of a viral pathogen.

Quantitative and Temporal Proteome Analysis of Butyrate-treated Colorectal Cancer Cells

Colorectal cancer is one of the most common cancers in developed countries, and its incidence is negatively associated with high dietary fiber intake. Butyrate, a short-chain fatty acid fermentation by-product of fiber induces cell maturation with the promotion of growth arrest, differentiation, and/or apoptosis of cancer cells. The stimulation of cell maturation by butyrate in colonic cancer cells follows a temporal progression from the early phase of growth arrest to the activation of apoptotic cascades. Previously we performed two-dimensional DIGE to identify differentially expressed proteins induced by 24-h butyrate treatment of HCT-116 colorectal cancer cells. Herein we used quantitative proteomics approaches using iTRAQ (isobaric tags for relative and absolute quantitation), a stable isotope labeling methodology that enables multiplexing of four samples, for a temporal study of HCT-116 cells treated with butyrate. In addition, cleavable ICAT, which selectively tags cysteine-containing proteins, was also used, and the results complemented those obtained from the iTRAQ strategy. Selected protein targets were validated by real time PCR and Western blotting. A model is proposed to illustrate our findings from this temporal analysis of the butyrate-responsive proteome that uncovered several integrated cellular processes and pathways involved in growth arrest, apoptosis, and metastasis. These signature clusters of butyrate-regulated pathways are potential targets for novel chemopreventive and therapeutic drugs for treatment of colorectal cancer.

Soluble amyloid β oligomers may contribute to apoptosis of retinal ganglion cells in glaucoma

Glaucoma is one of the leading causes of visual impairment and blindness. It is characterized by excavation of optic nerve head and visual field loss. Even though the pathogenesis of glaucoma remains unclear, it is generally accepted that elevated intraocular pressure is the major risk factor. No matter what the specific initiators are, retinal ganglion cells are believed to die via apoptosis eventually. It is known that glaucoma correlates strongly with Alzheimer's disease and the two diseases share many similarities in pathogenic mechanisms. Recent studies have indicated that amyloid beta peptide, which is implicated in the progression of Alzheimer's disease, may be also responsible for retinal ganglion cells death in glaucoma. Amyloid beta exists in different forms, including monomers, oligomers and fibrils, and among these, as demonstrated by extensive evidences, soluble amyloid beta oligomers rather than insoluble amyloid beta fibrils induced apoptosis of neurons in Alzheimer's disease. Here we propose that soluble amyloid beta oligomers may play an important role in activation of apoptotic cascades in retinal ganglion cells in glaucoma.

Vorinostat Synergistically Potentiates MK-0457 Lethality in Chronic Myelogenous Leukemia (CML) Cells Sensitive and Resistant to Imatinib Mesylate.

MK-0457/VX-680 was developed as a pan-aurora kinase inhibitor that induces apoptosis in tumor cells and suppresses tumor growth in vivo. Recently, it was found that MK-0457 is also a potent inhibitor of both wild type and mutated Bcr/Abl kinases, including the T315I mutant which confers resistance to first- (i.e., imatinib mesylate/IM) and second-generation kinase inhibitors (e.g., dasatinib and nilotinib). Moreover, MK-0457 exhibits significant activity in patients with T315I phenotype-refractory CML or Ph-positive ALL. Here we examined interactions between MK-0457 and the histone deacetylase inhibitor (HDACI) vorinostat/SAHA in Bcr/Abl+ leukemia cells, including those resistant to IM through various mechanisms, particularly the T315I mutation. Co-administration of non- or subtoxic concentrations of vorinostat (0.5–2 μM) increased the lethality of MK-0457 (5–100 nM) in a highly synergistic manner in K562 and LAMA84 cells. Similar interactions were observed in primary CD34+ CML cells, whereas the combination largely spared normal bone marrow mononuclear cells. Moreover, the MK-0457/vorinostat regimen markedly induced cell death in IM-resistant K562 cells exhibiting a Bcr/Abl-independent, Lyn-dependent form of resistance, a phenomenon accompanied by Lyn inactivation. Notably, the regimen was highly active against BaF/3 cells bearing various Bcr/Abl point mutations (i.e., T315I, E255K, and M351T) conferring resistance to IM. These events were associated with inactivation and downregulation of wild type and mutated Bcr/Abl (particularly T315I), reduced expression of phospho-CrkL, and vorinostat-mediated interruption of interactions between Bcr/Abl and Hsp90. Moreover, exposure of cells to marginally toxic doses of MK-0457 resulted in accumulation of cells with greater than 4N DNA content, resulting from induction of endoreduplication. Co-administration of vorinostat strikingly enhanced aurora kinase inhibition by MK-0457, reflected by markedly diminished expression of phospho-histone H3 (Ser 10), accompanied by preferential killing of polyploid cells. Interestingly, vorinostat also interacted with a selective inhibitor of aurora kinase A and B, at concentrations that inhibited histone H3 phosphorylation, to potentiate apoptosis in CML cells without modifying expression of phospho-Bcr/Abl or -CrkL. These results suggest that aurora kinase inhibition plays a functional role in synergistic interactions between HDACI and aurora kinase inhibitors. Finally, vorinostat markedly induced expression of the pro-apoptotic BH3-only protein Bim in various CML cell lines, including BaF/3 cells expressing the T315I Bcr/Abl mutation, as well as in primary CML cells. Knockdown of Bim by siRNA significantly protected K562 cells from apoptosis induced by vorinostat but not MK-0457. Furthermore, siRNA-mediated Bim downregulation blocked induction of Bim expression by vorinostat and dramatically diminished the capacity of this agent to potentiate MK-0457 lethality. In addition, blockade of apoptosis by Bim siRNA did not modify downregulation of Bcr/Abl or phospho-histone H3 in cells exposed to the vorinostat/MK-0457 regimen, arguing against the possibility that the latter phenomena are secondary to activation of apoptotic cascade. Together, these findings indicate that vorinostat dramatically increases MK-0457 lethality in IM-sensitive and -resistant CML cells, and suggest that inactivation of Bcr/Abl kinase and aurora kinases, as well as Bim induction by vorinostat, contribute to this phenomenon.

HSP70 STABILIZES THE LYSOSOMES BY TRANSPORTING MUC1 TO THE LYSOSOMES

We have previously shown that HSP70 is overexpressed in pancreatic cancer cells and human pancreatic cancer specimens and that inhibition of its expression leads to apoptotic cell death. Studies in our laboratory have also shown that inhibition of HSP70 expression leads lysosomal permeability with the activation of apoptotic cascade, thus suggesting that HSP70 stabilizes the lysosomes. However, the mechanism by which HSP70 stabilizes these two organelles and thus inhibits apoptosis is unclear. The aim ofthis study was to elucidate the role of MUC1, a mucin glycoprotein which is expressed in pancreatic cancer cells, in the stabilization of lysosomes and evaluating its interaction with HSP70. We hypothesize that HSP70 carries MUC1 to lysosomes and mitochondria which then stabilizes the lysosomes. Method: HSP70 and MUC1 were immunoprecipitated from the pancreatic cancer cell lysates. The immunoprecipitates where then immunoblotted for MUC1 and HSP70. To study the localization of MUC1 to lysosomes, co-localization of MUC1 and LAMP-2 (a lysosomal marker) was studied in pancreatic cancer cells by confocal microscopy. Effect of MUC1 knockdown by siRNA was studied by CCK-8 assay. Results: HSP70 and MUC1 co-immunoprecipitate suggesting their interaction. Confocal microscopy demonstrated co-localization of MUC1 and LAMP-1 suggesting that MUC1 localizes to lysosomes. Inhibition of MUC1 expression by siRNA leads to decreased viability in Panc-1 pancreatic cancer cells (54 ± 6 % of control treated with transfection reagent Lipofectamine 2000 only). Conclusion: the results suggest that MUC1 may play a role in the mechanism by which HSP70 stabilizes the lysosomes. This is the first report suggesting that MUC1 localizes to the lysosomes and may play a role in its stabilization. Implication: MUC1 may prove to be a potential target for the treatment of pancreatic cancer.

Histone deacetylase inhibitors induce apoptosis in both Type I and Type II endometrial cancer cells

Objective. To characterize the molecular pathways involved in apoptosis following administration of histone deacetylase inhibitors to Type I and II endometrial cancer cells. Methods. Ark2, Ishikawa, and AN3 cell lines representing both Type I and II endometrial cancers were treated with various concentrations of oxamflatin and HDAC inhibitor-1. Cell apoptosis was determined by flow cytometry, nuclear staining, Western blotting, and mitochondrial membrane potential assays. Results. Compared to controls, there was a 95% reduction in the growth of Ark2 cells following administration of histone deacetylase inhibitors and this response was dose-dependent. These agents also caused profound morphologic changes and loss of mitochondrial membrane potentials consistent with the induction of apoptosis. Cleavage of PARP, caspase-9, and caspase-8 was detected, confirming the activation of apoptotic cascades in endometrial carcinoma cells. This effect was present in both serous and endometrioid cell types. Conclusion. Our results suggest that oxamflatin and HDAC inhibitor-1 have potent cytotoxicity in endometrial cancer cells by inducing cell apoptosis. Histone deacetylase inhibitors are promising agents for the treatment of both Type I and II endometrial carcinoma.

Depolarization and cardiolipin depletion in aged rat brain mitochondria: Relationship with oxidative stress and electron transport chain activity

A noticeable loss of cardiolipin, a significant accumulation of fluorescent products of lipid peroxidation and an increased ability to produce reactive oxygen species in vitro are characteristics of aged rat brain mitochondria, as has been demonstrated in this study. In contrast mitochondrial electron transport chain activity is not significantly compromised except a marginal decline in complex IV activity in aged rat brain. On the other hand, a striking loss of mitochondrial membrane potential occurs in brain mitochondria during aging, which may be attributed to peroxidative membrane damage in this condition. Such mitochondrial dysfunctions as reported here may lead to uncoupling of oxidative phosphorylation, ATP depletion and activation of apoptotic cascade in aged rat brain.

Modulation of electron transport protects cardiac mitochondria and decreases myocardial injury during ischemia and reperfusion

Mitochondria are increasingly recognized as lynchpins in the evolution of cardiac injury during ischemia and reperfusion. This review addresses the emerging concept that modulation of mitochondrial respiration during and immediately following an episode of ischemia can attenuate the extent of myocardial injury. The blockade of electron transport and the partial uncoupling of respiration are two mechanisms whereby manipulation of mitochondrial metabolism during ischemia decreases cardiac injury. Although protection by inhibition of electron transport or uncoupling of respiration initially appears to be counterintuitive, the continuation of mitochondrial oxidative phosphorylation in the pathological milieu of ischemia generates reactive oxygen species, mitochondrial calcium overload, and the release of cytochrome c. The initial target of these deleterious mitochondrial-driven processes is the mitochondria themselves. Consequences to the cardiomyocyte, in turn, include oxidative damage, the onset of mitochondrial permeability transition, and activation of apoptotic cascades, all favoring cardiomyocyte death. Ischemia-induced mitochondrial damage carried forward into reperfusion further amplifies these mechanisms of mitochondrial-driven myocyte injury. Interruption of mitochondrial respiration during early reperfusion by pharmacologic blockade of electron transport or even recurrent hypoxia or brief ischemia paradoxically decreases cardiac injury. It increasingly appears that the cardioprotective paradigms of ischemic preconditioning and postconditioning utilize modulation of mitochondrial oxidative metabolism as a key effector mechanism. The initially counterintuitive approach to inhibit mitochondrial respiration provides a new cardioprotective paradigm to decrease cellular injury during both ischemia and reperfusion.

Role of Protein Conformational Dynamics and DNA Integrity in Relevance to Neuronal Cell Death in Neurodegeneration

Apoptosis has been implicated in the pathogenesis of various neurodegenerative disorders, although the extent to which it is responsible for the neurodegeneration along with other kind of cell death events is not known. Eventhough much information is available today on the apoptotic cascades in general, the precise mechanism and the exact sequence of events leading to neuronal degeneration in Alzheimer's disease (AD) and other neurodegenerative disorders is not understood till now. Amyloid beta (Abeta) proteins are the hallmark toxic proteins known to cause the activation of apoptotic cascades via caspase dependent and caspase-independent pathways. Abeta can cause neuronal apoptosis through multiple mechanisms involving mitochondria and endoplasmic reticulum as the key organelles. In this review, we have discussed the role of apoptosis in neurodegeneration and provided new thoughts on the role of protein conformational dynamics and DNA integrity associated with neurodegenerative disorders. An insight on whether the apoptosis observed in the neurodegenerative disorders is of any functional advantage has been discussed.

Increased susceptibility to amyloid toxicity in familial Alzheimer's fibroblasts

Much experimental evidence suggests that an imbalance in cellular redox status is a major factor in the pathogenesis of Alzheimer's disease (AD). Our previous data showed a marked increase in membrane lipoperoxidation in primary fibroblasts from familial AD (FAD) patients. In the present study, we demonstrate that when oligomeric structures of Aβ 1-40 and Aβ 1-42 are added to the culture media, they accumulate quicker near the plasma membrane, and are internalized faster and mostly in APPV717I fibroblasts than in age-matched healthy cells; this results in an earlier and sharper increase in the production of reactive oxygen species (ROS). Higher ROS production leads in turn to an increase in membrane oxidative-injury and significant impairment of cellular antioxidant capacity, giving rise to apoptotic cascade activation and finally to a necrotic outcome. In contrast, healthy fibroblasts appear more resistant to amyloid oxidative-attack, possibly as a result of their plasma membrane integrity and powerful antioxidant capacity. Our data are consistent with increasing evidence that prefibrillar aggregates, compared to mature fibrils, are likely the more toxic species of the peptides. These findings provide compelling evidence that cells bearing increased membrane lipoperoxidation are more susceptible to aggregate toxicity as a result of their reduced ability to counteract amyloid oligomeric attack.

Nitric oxide mediates increased susceptibility to dopaminergic damage in Nurr1 heterozygous mice

Knocking out of Nurr1 gene, a member of nuclear receptor superfamily, causes selective agenesis of dopaminergic neurons in midbrain. Reduced expression of Nurr1 increases the vulnerability of mesencephalic dopamine neurons to dopaminergic toxins. We evaluated the role of nitric oxide as a possible mechanism for this increased susceptibility. Increased expression of neuronal nitric oxide synthase and increased 3-nitrotyrosine were observed in striatum of Nurr1 heterozygous (Nurr1 +/-) mice as compared with wild-type. Increased cytochrome C activation and consecutive release of Smac/DIABLO were also observed in Nurr1 +/- mice. An induction of active Caspase-3 and p53, cleavage of poly-ADP (RNase) polymerase and reduced expression of bcl-2 were observed in Nurr1 +/- mice. Methamphetamine significantly increased these markers in Nurr1 +/- mice as compared with wild-type. The present data therefore suggest that nitric oxide plays a role as a modulating factor for the increased susceptibility, but not the potentiation, of the dopaminergic terminals in Nurr1 +/- mice. We also report that this increased neuronal nitric oxide synthase expression and increased nitration in Nurr1 +/- mice led to the activation of apoptotic cascade via the differential alterations in the DNA binding activity of transcription factors responsible for the propagation of growth arrest as well as apoptosis.

The Anti-Tumor Effect and Mechanisms of Action of Penta-Acetyl Geniposide

Gardenia, the fruit of Gardenia jasminoides Ellis, has been widely used to treat liver and gall bladder disorders in Chinese medicine. It has been shown recently that geniposide, the main ingredient of Gardenia Fructus, exhibits the anti-tumor effect. In this review, we discuss the anti-tumor effect and possible mechanisms of a derivative from Gardenia Fructus, penta-acetyl geniposide ((Ac)5GP). It has been demonstrated that (Ac)5GP plays more potent roles than geniposide in chemoprevention. (Ac)5GP decreased DNA damage and hepatocarcinogenesis induced by aflatoxin B1 (AFB1) by activating the phase II enzymes glutathione S-transferase (GST) and GSH peroxidase (GSH-Px). It reduced the growth and development of inoculated C6 glioma cells especially in pre-treated rats. In addition to the preventive effect, (Ac)5GP exerts its actions on apoptosis and growth arrest. Treatment of (Ac)5GP caused DNA fragmentation of glioma cells. (Ac)5GP induced sub- G1 peak through the activation of apoptotic cascades PKCdelta/JNK/Fas/caspase8 and caspase 3. Besides, p53/Bax signaling was suggested to be involved in (Ac)5GP-induced apoptosis, though its downstream cascades needs further clarified. (Ac)5GP has also been shown to inhibit DNA synthesis of tumor cells. It arrested cell cycle at G0/ G1 by inducing the expression of p21, thus suppressing the cyclin D1/cdk4 complex formation and the phosphorylation of E2F. The phosphorylation status of p53 on serine 392 correlated with the process of growth arrest. Evidences from the in vivo experiments showed that (Ac)5GP is not harmful to liver, heart and kidney. In conclusion, (Ac)5GP is highly suggested to be an anti-tumor agent for development in the future.

Mitochondrial Proteomic Analysis of a Cell Line Model of Familial Amyotrophic Lateral Sclerosis

Mutations in copper-zinc superoxide dismutase (SOD1) have been linked to a subset of familial amytrophic lateral sclerosis (fALS), a fatal neurodegenerative disease characterized by progressive motor neuron death. An increasing amount of evidence supports that mitochondrial dysfunction and apoptosis activation play a critical role in the fALS etiology, but little is known about the mechanisms by which SOD1 mutants cause the mitochondrial dysfunction and apoptosis. In this study, we use proteomic approaches to identify the mitochondrial proteins that are altered in the presence of a fALS-causing mutant G93A-SOD1. A comprehensive characterization of mitochondrial proteins from NSC34 cells, a motor neuron-like cell line, was achieved by two independent proteomic approaches. Four hundred seventy unique proteins were identified in the mitochondrial fraction collectively, 75 of which are newly discovered proteins that previously had only been reported at the cDNA level. Two-dimensional gel electrophoresis was subsequently used to analyze the differences between the mitochondrial proteomes of NSC34 cells expressing wild-type and G93A-SOD1. Nine and 36 protein spots displayed elevated and suppressed abundance respectively in G93A-SOD1-expressing cells. The 45 spots were identified by MS, and they include proteins involved in mitochondrial membrane transport, apoptosis, the respiratory chain, and molecular chaperones. In particular, alterations in the post-translational modifications of voltage-dependent anion channel 2 (VDAC2) were found, and its relevance to regulating mitochondrial membrane permeability and activation of apoptotic pathways is discussed. The potential role of other proteins in the mutant SOD1-mediated fALS is also discussed. This study has produced a short list of mitochondrial proteins that may hold the key to the mechanisms by which SOD1 mutants cause mitochondrial dysfunction and neuronal death. It has laid the foundation for further detailed functional studies to elucidate the role of particular mitochondrial proteins, such as VDAC2, in the pathogenesis of familial ALS.

Slight impairment of Na +,K +-ATPase synergistically aggravates ceramide- and β-amyloid-induced apoptosis in cortical neurons

Dysfunction of the Na +,K +-ATPase (Na +,K +-pump), due to reduced energy supply or increased endogenous ouabain-like inhibitors, likely occurs under pathological conditions in the central nervous system. In cultured mouse cortical neurons, we examined the hypothesis that a mild non-toxic inhibition of the Na +,K +-ATPase could synergistically sensitize the vulnerability of neurons to normally non-lethal apoptotic signals. Ouabain at a low concentration of 0.1 μM slightly lessened the Na +,K +-pump activity measured as an ouabain-sensitive current, yet did not affect K + homeostasis and viability of cortical neurons. Co-exposure to 0.1 μM ouabain plus non-lethal C 2-ceramide (5 μM) or β-amyloid 1–42 (5 μM), however, induced marked intracellular K + loss, caspase-3 cleavage, DNA laddering, and synergistically triggered neuronal death. The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) predominantly blocked the caspase activation and neuronal death. These results suggest that slight impairment of Na +,K +-pump activity may amplify the disruption of K + homeostasis in the presence of a non-lethal apoptotic insult, leading to activation of apoptotic cascade and substantial neuronal injury.