Translocation Of Apoptosis-inducing Factor Research Articles

Glyceraldehyde-3-phosphate Dehydrogenase Aggregates Accelerate Amyloid-β Amyloidogenesis in Alzheimer Disease

Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by loss of neurons and formation of pathological extracellular deposits induced by amyloid-β peptide (Aβ). Numerous studies have established Aβ amyloidogenesis as a hallmark of AD pathogenesis, particularly with respect to mitochondrial dysfunction. We have previously shown that glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forms amyloid-like aggregates upon exposure to oxidative stress and that these aggregates contribute to neuronal cell death. Here, we report that GAPDH aggregates accelerate Aβ amyloidogenesis and subsequent neuronal cell death both in vitro and in vivo. Co-incubation of Aβ40 with small amounts of GAPDH aggregates significantly enhanced Aβ40 amyloidogenesis, as assessed by in vitro thioflavin-T assays. Similarly, structural analyses using Congo red staining, circular dichroism, and atomic force microscopy revealed that GAPDH aggregates induced Aβ40 amyloidogenesis. In PC12 cells, GAPDH aggregates augmented Aβ40-induced cell death, concomitant with disruption of mitochondrial membrane potential. Furthermore, mice injected intracerebroventricularly with Aβ40 co-incubated with GAPDH aggregates exhibited Aβ40-induced pyramidal cell death and gliosis in the hippocampal CA3 region. These observations were accompanied by nuclear translocation of apoptosis-inducing factor and cytosolic release of cytochrome c from mitochondria. Finally, in the 3×Tg-AD mouse model of AD, GAPDH/Aβ co-aggregation and mitochondrial dysfunction were consistently detected in an age-dependent manner, and Aβ aggregate formation was attenuated by GAPDH siRNA treatment. Thus, this study suggests that GAPDH aggregates accelerate Aβ amyloidogenesis, subsequently leading to mitochondrial dysfunction and neuronal cell death in the pathogenesis of AD.

Rescue from Sexually Dimorphic Neuronal Cell Death by Estradiol and PI3 Kinase Activity.

Responses of primary hippocampal and cortical neurons derived from male and female rats to cellular stressors were studied. It is demonstrated that 17β-estradiol (E2), a potent neuroprotectant, protected the female neurons but had no effects on the male neurons from CoCl2- and glutamate-induced toxicity. Agonists of the estrogen receptor (ER) subtypes ERα and ERβ, DPN and PPT, respectively, had similar effects to E2. By contrast, effects of E2 were abolished by the ER antagonist ICI-182780, further corroborating the neuroprotective role of ERs. In male neurons, CoCl2 predominately activated the apoptosis-inducing factor (AIF)-dependent pathway and AIF translocation from the cytosol to the nucleus. In comparison, CoCl2 activated the caspase pathway and cytochrome c release in female neurons. The inhibitors of these pathways, namely DiQ for AIF and zVAD for caspase, specifically rescued CoCl2-induced cell death in male and female neurons, respectively. When zVAD and ICI-182780, and E2 were applied in combination, it was demonstrated E2 acted on the caspase pathway leading to female-specific neuroprotection. Furthermore, the PI3 kinase (PI3K) inhibitor blocked the rescue effects of DiQ and zVAD on the male and female neurons, respectively, suggesting that PI3K is a common upstream regulator for both pathways. The present study suggested that both sex-specific and nonspecific mechanisms played a role in neuronal responses to stressors and protective reagents.

FTY720 (Fingolimod) sensitizes hepatocellular carcinoma cells to sorafenib‐mediated cytotoxicity

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. The multityrosine kinase inhibitor sorafenib is used in the therapy of advanced disease. However, the effects of sorafenib are limited, and combination treatments aiming at improved survival are encouraged. The sphingosine analog FTY720 (Fingolimod), which is approved for treatment of multiple sclerosis, has shown tumor suppressive effects in cell lines and animal models of HCC. In the present study, we combined sorafenib with FTY720 in order to sensitize the HCC cell lines Huh7 and HepG2 to sorafenib treatment. Using the XTT assay we show that noncytotoxic doses of FTY720 synergistically enhanced the decrease in viability caused by treatment of both cell lines with increasing doses of sorafenib. Further studies in Huh7 revealed that combined treatment with FTY720 and sorafenib resulted in G1 arrest and enhanced cell death measured using flow cytometry analysis of cells labeled with propidium iodide (PI)/Annexin-V and PI and 4′,6-diamidino-2-phenylindole-staining of nuclei. In addition, signs of both caspase-dependent and – independent apoptosis were observed, as cotreatment with FTY720 and sorafenib caused cytochrome c release and poly-ADP ribose polymerase-cleavage as well as translocation of Apoptosis-inducing factor into the cytosol. We also detected features of autophagy blockage, as the protein levels of LC3-II and p62 were affected by combined treatment with FTY720 and sorafenib. Together, our results suggest that FTY720 sensitizes HCC cells to cytotoxic effects induced by treatment with sorafenib alone. These findings warrant further investigations of combined treatment with sorafenib and FTY720 in vivo in order to develop more effective treatment of HCC.

MicroRNA-345 induces apoptosis in pancreatic cancer cells through potentiation of caspase-dependent and -independent pathways.

Background:Previously, miR-345 was identified as one of the most significantly downregulated microRNAs in pancreatic cancer (PC); however, its functional significance remained unexplored.Methods:miR-345 was overexpressed in PC cells by stable transfection, and its effect on growth, apoptosis and mitochondrial-membrane potential was examined by WST-1, Hoechst-33342/Annexin-V, and JC-1 staining, respectively. Gene expression was examined by quantitative reverse-transcription-PCR and/or immunoblotting, and subcellular fractions prepared and caspase-3/7 activity determined by commercially available kits. miR-345 target validation was performed by mutational analysis and luciferase-reporter assay.Results:miR-345 is significantly downregulated in PC tissues and cell lines relative to normal pancreatic cells, and its expression decreases gradually in PC progression model cell lines. Forced expression of miR-345 results in reduced growth of PC cells because of the induction of apoptosis, accompanied by a loss in mitochondrial membrane potential, cytochrome-c release, caspases-3/7 activation, and PARP-1 cleavage, as well as mitochondrial-to-nuclear translocation of apoptosis-inducing factor. These effects could be reversed by the treatment of miR-345-overexpressing PC cells with anti-miR-345 oligonucleotides. BCL2 was characterised as a novel target of miR-345 and its forced-expression abrogated the effects of miR-345 in PC cells.Conclusions:miR-345 downregulation confers apoptosis resistance to PC cells, and its restoration could be exploited for therapeutic benefit.

LYG-202 exerts antitumor effect on PI3K/Akt signaling pathway in human breast cancer cells.

In this study, we aimed to investigate the antitumor effect of LYG-202, a newly synthesized piperazine-substituted derivative of flavonoid on human breast cancer cells and illustrate the potential mechanisms. LYG-202 induced apoptosis in MCF-7, MDA-MB-231 and MDA-MB-435 cells. LYG-202 triggered the activation of mitochondrial apoptotic pathway through multiple steps: increasing Bax/Bcl-2 ratio, decreasing mitochondrial membrane potential (ΔΨ(m)), activating caspase-9 and caspase-3, inducing cleavage of poly(ADP-ribose) polymerase, cytochrome c release and apoptosis-inducing factor translocation. Furthermore, LYG-202 inhibited cell cycle progression at the G1/S transition via targeting Cyclin D, CDK4 and p21(Waf1/Cip1). Additionally, LYG-202 increased the generation of intracellular ROS. N-Acetyl cysteine, an antioxidant, reversed LYG-202-induced apoptosis suggesting that LYG-202 induces apoptosis by accelerating ROS generation. Further, we found that LYG-202 deactivated the PI3K/Akt pathway, activated Bad phosphorylation, increased Cyclin D and Bcl-xL expression, and inhibited NF-κB nuclear translocation. Activation of PI3K/Akt pathway by IGF-1 attenuated LYG-202-induced apoptosis and cell cycle arrest. Our in vivo study showed that LYG-202 exhibited a potential antitumor effect in nude mice inoculated with MCF-7 tumor through similar mechanisms identified in cultured cells. In summary, our results demonstrated that LYG-202 induced apoptosis and cell cycle arrest via targeting PI3K/Akt pathway, indicating that LYG-202 is a potential anticancer agent for breast cancer.

Thioredoxin-dependent regulation of AIF-mediated DNA damage

The thioredoxin (Trx) system is one major redox system in mammalian cells. One of its component, Trx, is involved in redox homeostasis and many cellular biological processes through participating in disulfide reduction, S-nitrosylation/S-denitrosylation reactions and protein-protein interactions. In this study, we report the identification of a novel interaction between cytosolic/nuclear Trx1 and apoptosis inducing factor (AIF), and the redox sensitivity and biological significance of the Trx-AIF interaction was characterized. Cytosolic Trx1 but not mitochondrial Trx2 was observed to interact with AIF under physiological conditions and Trx1's active site cysteines were crucial for the interaction. Under oxidative stress conditions, Trx-AIF interaction was disrupted. When the treated cells were allowed to recover from oxidative stress by means of removal of the oxidants, interaction between Trx1 and AIF was re-established time-dependently, which underpins the biological relevance of a Trx-dependent redox regulation of AIF-mediated cell death. Indeed, in times of oxidative stress, nuclear translocation of AIF was found to occur concurrently with perturbations to the Trx-AIF interaction. Once localized in the nucleus, reduced Trx1 hindered the interaction between AIF and DNA, thereby bringing about an attenuation of AIF-mediated DNA damage. In conclusion, characterization of the Trx-AIF interaction has led to an understanding of the effect of reduced Trx1 on possibly regulating AIF-dependent cell death through impeding AIF-mediated DNA damage. Importantly, identification of the novel interaction between Trx1 and AIF has provided opportunities to design and develop therapeutically relevant strategies that either promote or prevent this protein-protein interaction for the treatment of different disease states.

Heat Shock Protein 70 Prevents Hyperoxia-Induced Disruption of Lung Endothelial Barrier via Caspase-Dependent and AIF-Dependent Pathways.

Exposure of pulmonary artery endothelial cells (PAECs) to hyperoxia results in a compromise in endothelial monolayer integrity, an increase in caspase-3 activity, and nuclear translocation of apoptosis-inducing factor (AIF), a marker of caspase-independent apoptosis. In an endeavor to identify proteins involved in hyperoxic endothelial injury, we found that the protein expression of heat-shock protein 70 (Hsp70) was increased in hyperoxic PAECs. The hyperoxia-induced Hsp70 protein expression is from hspA1B gene. Neither inhibition nor overexpression of Hsp70 affected the first phase barrier disruption of endothelial monolayer. Nevertheless, inhibition of Hsp70 by using the Hsp70 inhibitor KNK437 or knock down Hsp70 using siRNA exaggerated and overexpression of Hsp70 prevented the second phase disruption of lung endothelial integrity. Moreover, inhibition of Hsp70 exacerbated and overexpression of Hsp70 prevented hyperoxia-induced apoptosis, caspase-3 activation, and increase in nuclear AIF protein level in PAECs. Furthermore, we found that Hsp70 interacted with AIF in the cytosol in hyperoxic PAECs. Inhibition of Hsp70/AIF association by KNK437 correlated with increased nuclear AIF level and apoptosis in KNK437-treated PAECs. Finally, the ROS scavenger NAC prevented the hyperoxia-induced increase in Hsp70 expression and reduced the interaction of Hsp70 with AIF in hyperoxic PAECs. Together, these data indicate that increased expression of Hsp70 plays a protective role against hyperoxia-induced lung endothelial barrier disruption through caspase-dependent and AIF-dependent apoptotic pathways. Association of Hsp70 with AIF prevents AIF nuclear translocation, contributing to the protective effect of Hsp70 on hyperoxia-induced endothelial apoptosis. The hyperoxia-induced increase in Hsp70 expression and Hsp70/AIF interaction is contributed to ROS formation.

Protective effect of hydrogen-rich medium against high glucose-induced apoptosis of Schwann cells in vitro.

Diabetic peripheral neuropathy (DPN) is considered to be one of the most prevalent and life threatening microvascular diabetic complications. DPN affects up to 50% of patients with diabetes mellitus and there are currently no efficacious therapeutic strategies available for its treatment. Previous studies have reported that oxidative stress and poly(ADP‑ribose) polymerase‑1 (PARP‑1) may be unifying factors for hyperglycemic injury. The aim of the present study was to investigate the protective effects of hydrogen‑rich medium (HM) on high glucose (HG)‑mediated oxidative stress, PARP‑1 activation and the apoptosis of Schwann cells (SCs) in vitro. The cells were divided into different groups, and were treated for 48 h. Cell viability and apoptosis were evaluated using Cell Counting kit‑8 and annexin V/propidium iodide assays, respectively. The concentrations of 8‑hydroxy‑2‑deoxyguanosine (8‑OHdG) and peroxynitrite (ONOO‑) were detected using an enzyme‑linked immunosorbent assay. The presence of intracellular oxygen free radicals was confirmed using flow cytometric analysis. Colorimetric assays were performed to determine the activity of caspase‑3, and western blotting was performed to detect the protein expression levels of PARP‑1, cleaved PARP‑1, PAR, apoptosis‑inducing factor (AIF), B‑cell lymphoma 2 (Bcl‑2) and Bcl‑2‑associated X protein. HG was found to induce severe oxidative stress and promote the caspase‑dependent and caspase‑independent apoptosis of SCs. Treatment with HM inhibited HG‑induced oxidative stress by suppressing hydroxyl and ONOO‑ production, levels of 8‑OHdG, caspase‑3 activity and apoptosis in the SCs. Furthermore, treatment with HM downregulated the HG‑induced release of PAR, the activation of PARP‑1 and nuclear translocation of AIF, and upregulated the expression of Bcl‑2 in the SCs. These results indicated that HM inhibited the HG‑induced‑oxidative stress‑associated caspase‑dependent and caspase‑independent apoptotic pathways in SCs. Therefore, HM may have potential as a treatment for DPN.

Decreased proteasomal function accelerates cigarette smoke-induced pulmonary emphysema in mice

Chronic obstructive pulmonary disease (COPD) is a disease common in elderly people, characterized by progressive destruction of lung parenchyma and chronic inflammation of the airways. The pathogenesis of COPD remains unclear, but recent studies suggest that oxidative stress-induced apoptosis in alveolar cells contributes to emphysematous lung destruction. The proteasome is a multicatalytic enzyme complex that plays a critical role in proteostasis by rapidly destroying misfolded and modified proteins generated by oxidative and other stresses. Proteasome activity decreases with aging in many organs including lungs, and an age-related decline in proteasomal function has been implicated in various age-related pathologies. However, the role of the proteasome system in the pathogenesis of COPD has not been investigated. Recently, we have established a transgenic (Tg) mouse model with decreased proteasomal chymotrypsin-like activity, showing age-related phenotypes. Using this model, we demonstrate here that decreased proteasomal function accelerates cigarette smoke (CS)-induced pulmonary emphysema. CS-exposed Tg mice showed remarkable airspace enlargement and increased foci of inflammation compared with wild-type controls. Importantly, apoptotic cells were found in the alveolar walls of the affected lungs. Impaired proteasomal activity also enhanced apoptosis in cigarette smoke extract (CSE)-exposed fibroblastic cells derived from mice and humans in vitro. Notably, aggresome formation and prominent nuclear translocation of apoptosis-inducing factor were observed in CSE-exposed fibroblastic cells isolated from Tg mice. Collective evidence suggests that CS exposure and impaired proteasomal activity coordinately enhance apoptotic cell death in the alveolar walls that may be involved in the development and progression of emphysema in susceptible individuals such as the elderly.

Mitochondrial Division Inhibitor 1 Ameliorates Mitochondrial Injury, Apoptosis, and Motor Dysfunction After Acute Spinal Cord Injury in Rats.

Mitochondrial division inhibitor 1 (Mdivi-1) is the most effective pharmacological inhibitor of mitochondrial fission. Spinal cord injury (SCI) is a common and serious trauma, which lacks efficient treatment. This study aimed to detect the role of Mdivi-1 in neuronal injury and its underlying mechanism after acute SCI (ASCI) in rats. Western blot analysis showed that Bax levels on the mitochondrial outer membrane, and release of cytochrome C (cytC) and apoptosis-inducing factor (AIF) from the mitochondria began to increase significantly at 4h after ASCI, then peaked at 16h, and declined significantly from 16 to 24h. However, the mitochondrial levels of Bcl-2 increased significantly at 2h, peaked at 4h, and subsequently significantly decreased from 4 to 24h after ASCI. In addition, Mdivi-1(1.2mg/kg) significantly suppressed the translocation of dynamin-related protein 1 (Drp1) and Bax to the mitochondria, mitochondrial depolarization, decrease of ATP and reduced Glutathione, increase of the Malondialdehyde, cytC release, and AIF translocation at 16h and 3days after ASCI, and also inhibited the caspase-3 activation and decrease of the percentage of apoptotic cells at 16h, 3 and 10days, further, ameliorated the motor dysfunction greatly from 3 to 10days after ASCI in rats. This neuroprotective effect was dose-dependent. However, Mdivi-1(1.2mg/kg) had no effects on the translocation of Bcl-2 and fission protein 1 on the mitochondria, and did not affect the expression of total Drp1 at 16h after ASCI. Our experimental findings indicated that Mdivi-1 can protect rats against ASCI, and that its underlying mechanism may be associated with inhibition of Drp1 translocation to the mitochondria, alleviation of mitochondrial dysfunction and oxidative stress, and suppression of caspase-dependent and -independent apoptosis.

Calpain inhibitor PD150606 attenuates glutamate induced spiral ganglion neuron apoptosis through apoptosis inducing factor pathway in vitro.

ObjectiveThis research aimed to investigate whether glutamate induced spiral ganglion neurons (SGNs) apoptosis through apoptosis inducing factor (AIF) pathway. And verify whether PD150606, a calpain inhibitor could prevent apoptosis by inhibiting cleaving and releasing AIF in mitochondrion.MethodsSGNs of postnatal days 0-3 were harvested and cultured in dishes. 20 mM Glu, the caspase inhibitor Z-VAD-FMK and calpain inhibitor PD150606 were added into cultured dishes separately. We used optical microscope and immunofluoresence staining to observe cell morphology and AIF distribution, RT-PCR and Westernblot to analyse AIF and calpain expression in SGNs. TUNEL assay was used to test cell apoptosis.ResultsCell morphology and nuclear translocation of AIF were altered in SGNs by 20 mM Glu treated in vitro. The axon of SGN shortened, more apoptosis SGN were observed and the expression of AIF and calpain were up-regulated in Glu-treated group than the normal one (P<0.05). The same experiments were conducted in 20 mM+PD150606 treated group and 20 mM+Z-VAD-FMK group. Obviously AIF were located from cytoplasm to the nuclear and the expressions of AIF and calpain were down-regulated by PD150606 (P<0.05). Positive cells in TUNEL staining decreased after PD150606 treating. However, Z-VAD-FMK had no influence on AIF, calpain expression or cell apoptosis.ConclusionThe AIF-related apoptosis pathway is involved in the process of Glu-induced SGN injury. Furthermore, the inhibition of calpain can prevent AIF from releasing the nuclear or inducing SGN apoptosis.

Cigarette smoke causes caspase-independent apoptosis of bronchial epithelial cells from asthmatic donors.

BackgroundEpidemiologic studies have demonstrated important links between air pollution and asthma. Amongst these pollutants, environmental cigarette smoke is a risk factor both for asthma pathogenesis and exacerbation. As the barrier to the inhaled environment, the bronchial epithelium is a key structure that is exposed to cigarette smoke.ObjectivesSince primary bronchial epithelial cells (PBECs) from asthmatic donors are more susceptible to oxidant-induced apoptosis, we hypothesized that they would be susceptible to cigarette smoke-induced cell death.MethodsPBECs from normal and asthmatic donors were exposed to cigarette smoke extract (CSE); cell survival and apoptosis were assessed by fluorescence-activated cell sorting, and protective effects of antioxidants evaluated. The mechanism of cell death was evaluated using caspase inhibitors and immunofluorescent staining for apoptosis-inducing factor (AIF).ResultsExposure of PBEC cultures to CSE resulted in a dose-dependent increase in cell death. At 20% CSE, PBECs from asthmatic donors exhibited significantly more apoptosis than cells from non-asthmatic controls. Reduced glutathione (GSH), but not ascorbic acid (AA), protected against CSE-induced apoptosis. To investigate mechanisms of CSE-induced apoptosis, caspase-3 or -9 inhibitors were tested, but these failed to prevent apoptosis; in contrast, CSE promoted nuclear translocation of AIF from the mitochondria. GSH reduced the number of nuclear-AIF positive cells whereas AA was ineffective.ConclusionOur results show that PBECs from asthmatic donors are more susceptible to CSE-induced apoptosis. This response involves AIF, which has been implicated in DNA damage and ROS-mediated cell-death. Epithelial susceptibility to CSE may contribute to the impact of environmental tobacco smoke in asthma.

Annonaceous acetogenin mimic AA005 induces cancer cell death via apoptosis inducing factor through a caspase-3-independent mechanism.

BackgroundAnnonaceous acetogenins are a family of natural products with antitumor activities. Annonaceous acetogenin mimic AA005 reportedly inhibits mammalian mitochondrial NADH-ubiquinone reductase (Complex I) and induces gastric cancer cell death. However, the mechanisms underlying its cell-death-inducing activity are unclear.MethodsWe used SW620 colorectal adenocarcinoma cells to study AA005 cytotoxic activity. Cell deaths were determined by Trypan blue assay and flow cytometry, and related proteins were characterized by western blot. Immunofluorescence and subcellular fractionation were used to evaluate AIF nuclear translocation. Reactive oxygen species were assessed by using redox-sensitive dye DCFDA.ResultsAA005 induces a unique type of cell death in colorectal adenocarcinoma cells, characterized by lack of caspase-3 activation or apoptotic body formation, sensitivity to poly (ADP-ribose) polymerase inhibitor Olaparib (AZD2281) but not pan-caspase inhibitor Z-VAD.fmk, and dependence on apoptosis-inducing factor (AIF). AA005 treatment also reduced expression of mitochondrial Complex I components, and leads to accumulation of intracellular reactive oxygen species (ROS) at the early stage. Blocking ROS formation significantly suppresses AA005-induced cell death in SW620 cells. Moreover, blocking activation of RIP-1 by necroptosis inhibitor necrotatin-1 inhibits AIF translocation and partially suppresses AA005-induced cell death in SW620 cells demonstrating that RIP-1 protein may be essential for cell death.ConclusionsAA005 may trigger the cell death via mediated by AIF through caspase-3 independent pathway. Our work provided new mechanisms for AA005-induced cancer cell death and novel clues for cancer treatment via AIF dependent cell death.

Cyclosporine A increases hair follicle growth by suppressing apoptosis-inducing factor nuclear translocation: a new mechanism.

Cyclosporine A (CsA) enhances hair growth through caspase-dependent pathways by retarding anagen-to-catagen phase transition in the hair follicle growth cycle. Whether apoptosis-inducing factor (AIF), a protein that induces caspase-independent apoptosis, can regulate the hair follicle cycle in response to CsA is currently unclear. Here, we show that the pro-hair growth properties of CsA are in part due to blockage of AIF nuclear translocation. We first isolate hair follicles from murine dorsal skin. We then used Western blot, immunohistochemistry and immunofluorescence to evaluate the expression and localization of AIF in hair follicles. We also determined whether modulation of AIF was responsible for the effects of CsA at the anagen-to-catagen transition. AIF was expressed in hair follicles during the anagen, catagen and telogen phases. There was significant nuclear translocation of AIF as hair follicles transitioned from anagen to late catagen phase; this was inhibited by CsA, likely due to reduced cyclophilin A expression and attenuated AIF release from mitochondria. However, we note that AIF translocation was not completely eliminated, which likely explains why the transition to catagen phase was severely retarded by CsA, rather than being completely inhibited. We speculate that blockade of the AIF signalling pathway is a critical event required for CsA-dependent promotion of hair growth in mice. The study of AIF-related signalling pathways may provide insight into hair diseases and suggest potential novel therapeutic strategies.

Cathepsin K knockout alleviates aging-induced cardiac dysfunction.

Aging is a major risk factor for cardiovascular disease. It has previously been shown that protein levels of cathepsin K, a lysosomal cysteine protease, are elevated in the failing heart and that genetic ablation of cathepsin K protects against pressure overload-induced cardiac hypertrophy and contractile dysfunction. Here we test the hypothesis that cathepsin K knockout alleviates age-dependent decline in cardiac function. Cardiac geometry, contractile function, intracellular Ca2+ properties, and cardiomyocyte apoptosis were evaluated using echocardiography, fura-2 technique, immunohistochemistry, Western blot and TUNEL staining, respectively. Aged (24-month-old) mice exhibited significant cardiac remodeling (enlarged chamber size, wall thickness, myocyte cross-sectional area, and fibrosis), decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca2+ release compared to young (6-month-old) mice, which were attenuated in the cathepsin K knockout mice. Cellular markers of senescence, including cardiac lipofuscin, p21 and p16, were lower in the aged-cathepsin K knockout mice compared to their wild-type counterpart. Mechanistically, cathepsin K knockout mice attenuated an age-induced increase in cardiomyocyte apoptosis and nuclear translocation of mitochondrial apoptosis-inducing factor (AIF). In cultured H9c2 cells, doxorubicin stimulated premature senescence and apoptosis. Silencing of cathepsin K blocked the doxorubicin-induced translocation of AIF from the mitochondria to the nuclei. Collectively, these results suggest that cathepsin K knockout attenuates age-related decline in cardiac function via suppressing caspase-dependent and caspase-independent apoptosis.

Carbon ion beam triggers both caspase-dependent and caspase-independent pathway of apoptosis in HeLa and status of PARP-1 controls intensity of apoptosis.

High linear energy transfer (LET) carbon ion beam (CIB) is becoming very promising tool for various cancer treatments and is more efficient than conventional low LET gamma or X-rays to kill malignant or radio-resistant cells, although detailed mechanism of cell death is still unknown. Poly (ADP-ribose) polymerase-1 (PARP-1) is a key player in DNA repair and its inhibitors are well-known as radio-sensitizer for low LET radiation. The objective of our study was to find mechanism(s) of induction of apoptosis by CIB and role of PARP-1 in CIB-induced apoptosis. We observed overall higher apoptosis in PARP-1 knocked down HeLa cells (HsiI) compared with negative control H-vector cells after irradiation with CIB (0-4Gy). CIB activated both intrinsic and extrinsic pathways of apoptosis via caspase-9 and caspase-8 activation respectively, followed by caspase-3 activation, apoptotic body, nucleosomal ladder formation and sub-G1 accumulation. Apoptosis inducing factor translocation into nucleus in H-vector but not in HsiI cells after CIB irradiation contributed caspase-independent apoptosis. Higher p53 expression was observed in HsiI cells compared with H-vector after exposure with CIB. Notably, we observed about 37% fall of mitochondrial membrane potential, activation of caspase-9 and caspase-3 and mild activation of caspase-8 without any detectable apoptotic body formation in un-irradiated HsiI cells. We conclude that reduction of PARP-1 expression activates apoptotic signals via intrinsic and extrinsic pathways in un-irradiated cells. CIB irradiation further intensified both intrinsic and extrinsic pathways of apoptosis synergistically along with up-regulation of p53 in HsiI cells resulting overall higher apoptosis in HsiI than H-vector.

NAD+ treatment can prevent rotenone-induced increases in DNA damage, Bax levels and nuclear translocation of apoptosis-inducing factor in differentiated PC12 cells.

Nicotinamide adenine dinucleotide (NAD(+)) plays critical roles in energy metabolism, mitochondrial functions, calcium homeostasis and immunological functions. Our previous studies have found that NAD(+) administration can profoundly decrease ischemic brain injury and traumatic brain injury. Our recent study has also provided first direct evidence indicating that NAD(+) treatment can decrease cellular apoptosis, while the mechanisms underlying this protective effect remain unclear. In our current study, we determined the effects of NAD(+) treatment on several major factors in apoptosis and necrosis, including levels of Bax and nuclear translocation of apoptosis-inducing factor (AIF), as well as levels of DNA double-strand breaks (DSBs) and intracellular ATP in rotenone-treated differentiated PC12 cells. We found that NAD(+) treatment can markedly attenuate the rotenone-induced increases in the levels of Bax and nuclear translocation of AIF in the cells. We further found that NAD(+) treatment can significantly attenuate the rotenone-induced increase in the levels of DSBs and decrease in the intracellular ATP levels. Collectively, our study has suggested mechanisms underlying the preventive effects of NAD(+) on apoptosis, which has highlighted the therapeutic potential of NAD(+) for decreasing apoptotic changes in multiple major diseases.

Valproic acid induces neuronal cell death through a novel calpain-dependent necroptosis pathway.

A growing body of evidence indicates that valproic acid (VPA), a histone deacetylase inhibitor used to treat epilepsy and mood disorders, has histone deacetylase-related and -unrelated neurotoxic activity, the mechanism of which is still poorly understood. We report that VPA induces neuronal cell death through an atypical calpain-dependent necroptosis pathway that initiates with downstream activation of c-Jun N-terminal kinase 1 (JNK1) and increased expression of receptor-interacting protein 1 (RIP-1) and is accompanied by cleavage and mitochondrial release/nuclear translocation of apoptosis-inducing factor, mitochondrial release of Smac/DIABLO, and inhibition of the anti-apoptotic protein X-linked inhibitor of apoptosis (XIAP). Coinciding with apoptosis-inducing factor nuclear translocation, VPA induces phosphorylation of the necroptosis-associated histone H2A family member H2AX, which is known to contribute to lethal DNA degradation. These signals are inhibited in neuronal cells that express constitutively activated MEK/ERK and/or PI3-K/Akt survival pathways, allowing them to resist VPA-induced cell death. The data indicate that VPA has neurotoxic activity and identify a novel calpain-dependent necroptosis pathway that includes JNK1 activation and RIP-1 expression. A growing body of evidence indicates that valproic acid (VPA) has neurotoxic activity, the mechanism of which is still poorly understood. We report, for the first time, that VPA activates a previously unrecognized calpain-dependent necroptosis cascade that initiates with JNK1 activation and involves AIF cleavage/nuclear translocation and H2AX phosphorylation as well as an altered Smac/DIABLO to XIAP balance.

Neuroprotective effects of mGluR II and III activators against staurosporine- and doxorubicin-induced cellular injury in SH-SY5Y cells: New evidence for a mechanism involving inhibition of AIF translocation

There are several experimental data sets demonstrating the neuroprotective effects of activation of group II and III metabotropic glutamate receptors (mGluR II/III), however, their effect on neuronal apoptotic processes has yet to be fully recognized. Thus, the comparison of the neuroprotective potency of the mGluR II agonist LY354740, mGluR III agonist ACPT-I, mGluR4 PAM VU0361737, mGluR8 PAM AZ12216052 and allosteric mGluR7 agonist AMN082 against staurosporine (St-) and doxorubicin (Dox)-induced cell death has been performed in undifferentiated (UN-) and retinoic acid differentiated (RA-) human neuroblastoma SH-SY5Y cells. The highest neuroprotection in UN-SH-SY5Y cells was noted for AZ12216052 (0.01–1 µM) and VU0361737 (1–10 µM), with both agents partially attenuating the St- and Dox-evoked cell death. LY354740 (0.01–10 µM) and ACPT-I (10 µM) were protective only against the St-evoked cell damage, whereas AMN082 (0.001–0.01 µM) attenuated only the Dox-induced cell death. In RA-SH-SY5Y, a moderate neuroprotective response of mGluR II/III activators was observed for LY354740 (10 µM) and AZ12216052 (0.01 and 10 µM), which afforded protection only against the St-induced cell damage. The protection mediated by mGluR II/III activators against the St- and Dox-evoked cell death in UN-SH-SY5Y cells was not related to attenuation of caspase-3 activity, however, a decrease in the number of TUNEL-positive nuclei was found. Moreover, mGluR II/III activators attenuated the cytosolic level of the apoptosis inducing factor (AIF), which was increased after St and Dox exposure. Our data point to differential neuroprotective efficacy of various mGluR II/III activators in attenuating St- and Dox-evoked cell damage in SH-SY5Y cells, and dependence of the effects on the cellular differentiation state, as well on the type of the pro-apoptotic agent that is employed. Moreover, the neuroprotection mediated by mGluR II/III activators is accompanied by inhibition of caspase-3-independent DNA fragmentation evoked by AIF translocation.

Cell death pathway induced by resveratrol-bovine serum albumin nanoparticles in a human ovarian cell line.

Resveratrol-bovine serum albumin nanoparticles (RES-BSANP) exhibit chemotherapeutic properties, which trigger apoptosis. The aim of the present study was to investigate the caspase-independent cell death pathway induced by RES-BSANP in human ovarian cancer SKOV3 cells and to analyze its mechanism. Morphological changes were observed by apoptotic body/cell nucleus DNA staining using inverted and fluorescence microscopy. The cell death pathway was determined by phosphatidylserine translocation. Western blot analysis was conducted to detect the activation of apoptosis-inducing factor (AIF), cytochrome c (Cyto c) and B-cell lymphoma 2-associated X protein (Bax). Apoptotic body and nuclear condensation and fragmentation were observed simultaneously following treatment with RES-BSANP. RES-BSANP induced apoptosis in a dose-dependent manner in the human ovarian cancer SKOV3 cells. The translocation of AIF from the mitochondria to the cytoplasm occurred earlier than that of Cyto c. In addition, Bax binding to the mitochondria was required for the release of AIF and Cyto c from the mitochondria. The AIF apoptosis pathway may present an alternative caspase-dependent apoptosis pathway in human ovarian cell death induced by RES-BSANP. Elucidation of this pathway may be critical for the treatment of cancer using high doses of RES-BSANP.