Translocation Of Apoptosis-inducing Factor Research Articles

P53- and ROS-mediated AIF pathway involved in TGEV-induced apoptosis.

We previously demonstrated that transmissible gastroenteritis virus (TGEV) could induce apoptosis through caspase signaling. However, apoptosis was not completely prevented by caspases inhibitors, suggesting that there may be a caspase-independent pathway involved in TGEV-induced cell apoptosis. In this study, we investigated the regulation of apoptosis-inducing factor (AIF) on TGEV-induced apoptotic pathway. Results indicated that AIF translocated from the mitochondria to nucleus during TGEV infection, and the AIF inhibitor, N-phenylmaleimide (NP), significantly attenuated the apoptosis. In addition, the translocation of AIF was inhibited by Veliparib (ABT-888), an inhibitor of poly (ADP-ribose) polymerase (PARP). And the reactive oxygen species (ROS) scavenger, pyrrolidinedithiocarbamic (PDTC), redistributed AIF in the mitochondria and nucleus in TGEV-infected cells. Moreover, the protein levels in nucleus and the mRNA levels of AIF were inhibited in the presence of the p53 inhibitor, pifithrin-α (PFT-α) or in TGEV-infected p53−/−cells. Furthermore, TGEV-induced apoptosis was blocked by combination of three or more inhibitors, such as pan caspase inhibitor Z-VAD-FMK, NP, ABT-888, PDTC, PFT-α, to treat PK-15 cells. Taken together, these results suggest that the p53- and ROS-mediated AIF pathway and caspase-dependent pathway were involved in TGEV-induced apoptosis.

Hydrogen peroxide induces programmed necrosis in rat nucleus pulposus cells through the RIP1/RIP3-PARP-AIF pathway.

This study aimed to systematically investigate whether programmed necrosis contributes to H2 O2 -induced nucleus pulposus (NP) cells death and to further explore the underlying mechanism involved. Rat NP cells were subjected to different concentrations of H2 O2 for various time periods. The cell viability was measured using a cell counting kit-8, and the death rate was detected by Hoechst 33258/propidium iodide (PI) staining. The programmed necrosis-related molecules receptor-interacting protein 1 (RIP1), receptor-interacting protein 3 (RIP3), poly (ADP-ribose) polymerase (PARP), and apoptosis inducing factor (AIF) were determined by real-time polymerase chain reaction and Western blotting, respectively. The morphologic and ultrastructural changes were examined by phasecontrast microscopy and transmission electron microscopy (TEM). In addition, the necroptosis inhibitor Necrostatin-1 (Nec-1), the PARP inhibitor diphenyl-benzoquinone (DPQ) and small interfering RNA (siRNA) technology were used to indirectly evaluate programmed necrosis. Our results indicated that H2 O2 induced necrotic morphologic and ultrastructural changes and an elevated PI positive rate in NP cells; these effects were mediated by the upregulation of RIP1 and RIP3, hyperactivation of PARP, and translocation of AIF from mitochondria to nucleus. Additionally, NP cells necrosis was significantly attenuated by Nec-1, DPQ pretreatment and knockdown of RIP3 and AIF, while knockdown of RIP1 produced the opposite effects. In conclusion, these results suggested that under oxidative stress, RIP1/RIP3-mediated programmed necrosis, executed through the PARP-AIF pathway, played an important role in NP cell death. Protective strategies aiming to regulate programmed necrosis may exert a beneficial effect for NP cells survival, and ultimately retard intervertebral disc (IVD) degeneration. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1269-1282, 2018.

Calpain and JNK pathways participate in isoflurane – induced nucleus translocation of apoptosis-inducing factor in the brain of neonatal rats

Recent studies have demonstrated that volatile anesthetic causes caspase-dependent neuroapoptosis and persistent cognitive deficits in young animals. Apoptosis-inducing factor (AIF) can trigger apoptosis by caspase-independent pathway. Whether isoflurane induces neuroapoptosis by activation of AIF and its possible mechanism are underdetermined. Rats at postnatal day 7 were exposed to 1.1% isoflurane for 4 h and the expression of AIF, cytochrome c, caspase-3, μ-calpain, m-calpain, Bcl-2 and Bax in the mitochondrial, cytosolic, and nuclear fraction, as well as the number of both AIF and TUNEL positive neurons in the cortices of rats were measured. Moreover, the effects of calpain inhibitor MDL-28170 or JNK inhibitor SP600125 on isoflurane-induced AIF release, caspase activation and cognitive deficits were assessed. We found isoflurane activated CytC-caspase-3 dependent apoptosis pathway mainly in the early phase (0–6 h after exposure). Moreover, isoflurane activated mitochondrial μ-calpain, induced AIF truncation during early phase and activated m-calpain, induced AIF release from the mitochondria to cytosol and translocation into the nucleus in the late phase (6–24 h after exposure). MDL-28170 attenuated the isoflurane–induced mitochondrial AIF truncation, release and nuclear translocation, but did not change the expression of cleaved-caspase-3 and mitochondrial Bax and Bcl-2 proteins. SP600125 attenuated isoflurane-induced neuroapoptosis by inhibiting both AIF and caspase-3 pathways and reduced cognitive impairment in neonatal rats. This is the first study to provide the evidence that isoflurane induced AIF-dependent neuroapoptosis by activation of mitochondrial μ-calpain and m-calpain in neonatal rats. JNK inhibition reversed isoflurane-induced neuroapoptosis and subsequent long-term neurocognitive impairment, acting via inhibiting activation of both AIF and caspase-3 pathways.

Mild hypothermia protects neurons against oxygen glucose deprivation via poly (ADP-ribose) signaling

Objective: Hypothermia is a neuroprotective mechanism that has been validated for use in alleviating neonatal hypoxic-ischemic (HI) brain injury. Nevertheless, it is unclear whether poly (ADP-ribose) (PAR) signaling is involved in hypothermia-induced neuroprotection. In this study, we investigated whether mild hypothermia rescues oxygen glucose deprivation (OGD)-induced cell death by modifying PAR-relative protein expression, such as AIF, PARP-1, and PAR polymer, in primary-cultured hippocampal neurons.Methods: We analyzed neuronal morphology and related protein expression of PAR signaling after OGD followed by mild hypothermia in primary-cultured newborn hippocampal neurons.Results: Hypothermic treatment resulted in improved neuronal viability and alleviated DNA damage. Results from the protein assay showed that hypothermia attenuated nuclear translocation of apoptosis-inducing factor (AIF), inhibited overactivation of poly(ADP-ribose) polymerase-1 (PARP-1), and decreased production of PAR polymer induced by PARP-1 activation after OGD.Conclusions: These results showed that mild hypothermia partially protects immature hippocampal neurons against OGD injury in part by interfering with the PAR signaling pathway.

Brain- and brain tumor-penetrating disulfiram nanoparticles: Sequence of cytotoxic events and efficacy in human glioma cell lines and intracranial xenografts

There is great interest in repurposing disulfiram (DSF), a rapidly metabolizing nontoxic drug, for brain cancers and other cancers. To overcome the instability and low therapeutic efficacy, we engineered passively-targeted DSF-nanoparticles (DSFNPs) using biodegradable monomethoxy (polyethylene glycol) d,l-lactic-co-glycolic acid (mPEG-PLGA) matrix. The physicochemical properties, cellular uptake and the blood brain-barrier permeability of DSFNPs were investigated. The DSFNPs were highly stable with a size of ∼70 nm with a >90% entrapment. Injection of the nanoparticles labeled with HITC, a near-infrared dye into normal mice and tumor-bearing nude mice followed by in vivo imaging showed a selective accumulation of the formulation within the brain and subcutaneous tumors for >24 h, indicating an increased plasma half-life and entry of DSF into desired sites. The DSFNPs induced a potent and preferential killing of many brain tumor cell lines in cytotoxicity assays. Confocal microscopy showed a quick internalization of the nanoparticles in tumor cells followed by initial accumulation in lysosomes and subsequently in mitochondria. DSFNPs induced high levels of ROS and led to a marked loss of mitochondrial membrane potential. Activation of the MAP-kinase pathway leading to a nuclear translocation of apoptosis-inducing factor and altered expression of apoptotic and anti-apoptotic proteins were also observed. DSFNPs induced a powerful and significant regression of intracranial medulloblastoma xenografts compared to the marginal efficacy of unencapsulated DSF. Together, we show that passively targeted DSFNPs can affect multiple targets, trigger potent anticancer effects, and can offer a sustained drug supply for brain cancer treatment through an enhanced permeability retention (EPR).

The sirtuin 1/2 inhibitor tenovin-1 induces a nonlinear apoptosis-inducing factor-dependent cell death in a p53 null Ewing's sarcoma cell line.

The sirtuin 1/2 inhibitor tenovin-1 activates p53 and may have potential in the management of cancer. Here, we investigated the responsiveness of Ewing's sarcoma cells to tenovin-1. We examined its effects in two Ewing's sarcoma cell lines with different p53 status, i.e. in p53 wild-type and p53 null cells. Effects were assessed by flow cytometric analyses of cell death, mitochondrial membrane depolarization and reactive oxygen species (ROS) generation, by caspase 3/7 activity measurement, by mRNA expression profiling and by immunoblotting. Tenovin-1 elicited caspase-mediated cell death in p53 wild-type cells, but caspase-independent cell death in p53 null cells. Remarkably, it induced a nonlinear concentration response in the latter: low concentrations of tenovin-1 were much more effective than were higher concentrations. Tenovin-1's effects in p53 null cells involved gene expression changes of Bcl-2 family members, mitochondrial membrane depolarization, nuclear translocation of apoptosis-inducing factor, ROS formation and DNA damage; all these effects followed a bell-shaped pattern. In conclusion, our results provide new insights into tenovin-1's mode of action by demonstrating that it can induce different pathways of cell death.

Bid-Induced Release of AIF/EndoG from Mitochondria Causes Apoptosis of Macrophages during Infection with Leptospira interrogans.

Leptospirosis is a global zoonotic infectious disease caused by pathogenic Leptospira species. Leptospire-induced macrophage apoptosis through the Fas/FasL-caspase-8/3 pathway plays an important role in the survival and proliferation of the pathogen in hosts. Although, the release of mitochondrial apoptosis-inducing factor (AIF) and endonuclease G (EndoG) in leptospire-infected macrophages has been described, the mechanisms linking caspase and mitochondrion-related host-cell apoptosis has not been determined. Here, we demonstrated that leptospire-infection induced apoptosis through mitochondrial damages in macrophages. Apoptosis was caused by the mitochondrial release and nuclear translocation of AIF and/or EndoG, leading to nuclear DNA fragmentation. However, the mitochondrion-related CytC-caspase-9/3 pathway was not activated. Next, we found that the release and translocation of AIF and/or EndoG was preceded by the activation of the BH3-interacting domain death agonist (Bid). Furthermore, our data demonstrated that caspase-8 was activated during the infection and caused the activation of Bid. Meanwhile, high reactive oxygen species (ROS) trigged by the infection caused the dephosphorylation of Akt, which also activated Bid. In conclusion, Bid-mediated mitochondrial release of AIF and/or EndoG followed by nuclear translocation is a major mechanism of leptospire- induced apoptosis in macrophages, and this process is modulated by both caspase-8 and ROS-Akt signal pathways.

Kinase-independent role of nuclear RIPK1 in regulating parthanatos through physical interaction with PARP1 upon oxidative stress

Regulated necrosis occurs in various pathophysiological conditions under oxidative stress. Here, we report that receptor-interacting protein kinase 1 (RIPK1), a key player in one type of regulated necrosis (necroptosis), also participates in another type of poly (ADP-ribose) polymerase 1 (PARP1)-dependent regulated necrosis (parthanatos). Various biological signatures of parthanatos were significantly attenuated in Ripk1−/− mouse embryonic fibroblasts, including PARylation, nuclear translocation of apoptosis-inducing factor, and PARP1-dependent cell death under H2O2 exposure. Hence, we investigated whether RIPK1 regulates the activity of PARP1. RIPK1 activated PARP1 via an interaction with the catalytic domain of PARP1 in the nucleus. Of note, both wild type and kinase-dead mutant RIPK1 induced PARP1 activation and led to PARP1-mediated cell death upon H2O2 insult, demonstrating the kinase-independent regulation of RIPK1 in PARP1 activation. Collectively, our results demonstrate the existence of a kinase-independent role of nuclear RIPK1 in the regulation of PARP1.

The dual role of poly(ADP-ribose) polymerase-1 in modulating parthanatos and autophagy under oxidative stress in rat cochlear marginal cells of the stria vascularis

Oxidative stress is reported to regulate several apoptotic and necrotic cell death pathways in auditory tissues. Poly(ADP-ribose) polymerase-1 (PARP-1) can be activated under oxidative stress, which is the hallmark of parthanatos. Autophagy, which serves either a pro-survival or pro-death function, can also be stimulated by oxidative stress, but the role of autophagy and its relationship with parthanatos underlying this activation in the inner ear remains unknown. In this study, we established an oxidative stress model in vitro by glucose oxidase/glucose (GO/G), which could continuously generate low concentrations of H2O2 to mimic continuous exposure to H2O2 in physiological conditions, for investigation of oxidative stress-induced cell death mechanisms and the regulatory role of PARP-1 in this process. We observed that GO/G induced stria marginal cells (MCs) death via upregulation of PARP-1 expression, accumulation of polyADP-ribose (PAR) polymers, decline of mitochondrial membrane potential (MMP) and nuclear translocation of apoptosis-inducing factor (AIF), which all are biochemical features of parthanatos. PARP-1 knockdown rescued GO/G-induced MCs death, as well as abrogated downstream molecular events of PARP-1 activation. In addition, we demonstrated that GO/G stimulated autophagy and PARP-1 knockdown suppressed GO/G-induced autophagy in MCs. Interestingly, autophagy suppression by 3-Methyladenine (3-MA) accelerated GO/G-induced parthanatos, indicating a pro-survival function of autophagy in GO/G-induced MCs death. Taken together, these data suggested that PARP-1 played dual roles by modulating parthanatos and autophagy in oxidative stress-induced MCs death, which may be considered as a promising therapeutic target for ameliorating oxidative stress-related hearing disorders.

Novel histone deacetylase inhibitor AR-42 exhibits antitumor activity in pancreatic cancer cells by affecting multiple biochemical pathways.

ObjectivePancreatic cancer is one of the most lethal types of cancer with a 5-year survival rate of ~5%. Histone deacetylases (HDACs) participate in many cellular processes, including carcinogenesis, and pharmacological inhibition of HDACs has emerged as a potential therapeutic strategy. In this study, we explored antitumor activity of the novel HDAC inhibitor AR-42 in pancreatic cancer.MethodsHuman pancreatic cancer cell lines BxPC-3 and PANC-1 were used in this study. Real-time PCR, RT-PCR, and western blotting were employed to investigate expression of specific genes and proteins, respectively. Translocation of apoptosis-inducing factor was investigated by immunofluorescence and subcellular fractionation. The number of apoptotic cells, cell cycle stages, and reactive oxygen species (ROS) generation levels were determined by flow cytometry. Cell invasiveness was examined by the Matrigel invasion assay. Efficacy of AR-42 in vivo was evaluated by utilizing BxPC-3 xenograft mouse model.ResultsAR-42 inhibited pancreatic cancer cell proliferation by causing G2/M cell cycle arrest via regulating expression levels of genes and proteins involved in cell cycle. AR-42 also induced ROS generation and DNA damage, triggering apoptosis of pancreatic cancer cells via both caspase-3-dependent and caspase-3-independent pathways. In addition, AR-42 increased expression levels of negative regulators of p53 (miR-125b, miR-30d, and miR33), which could contribute to lower expression level of mutant p53 in pancreatic cancer cells. Cell invasion assay showed that AR-42 reduced cancer cell aggressiveness and significantly diminished BxPC-3 xenograft tumor growth in vivo.ConclusionAR-42, a novel HDAC inhibitor, inhibited pancreatic cancer cells by regulating p53 expression, inducing cell cycle arrest, particularly at the G2/M stage, and activating multiple apoptosis pathways. Additionally, AR-42 inhibited cell invasiveness and potently suppressed pancreatic cancer tumors in vivo. We conclude that by virtue of its multiple mechanisms of action, AR-42 possesses a considerable potential as an antitumor agent in pancreatic cancer.

The erythropoietin-derived peptide MK-X and erythropoietin have neuroprotective effects against ischemic brain damage

Erythropoietin (EPO) has been well known as a hematopoietic cytokine over the past decades. However, recent reports have demonstrated that EPO plays a neuroprotective role in the central nervous system, and EPO has been considered as a therapeutic target in neurodegenerative diseases such as ischemic stroke. Despite the neuroprotective effect of EPO, clinical trials have shown its unexpected side effects, including undesirable proliferative effects such as erythropoiesis and tumor growth. Therefore, the development of EPO analogs that would confer neuroprotection without adverse effects has been attempted. In this study, we examined the potential of a novel EPO-based short peptide, MK-X, as a novel drug for stroke treatment in comparison with EPO. We found that MK-X administration with reperfusion dramatically reduced brain injury in an in vivo mouse model of ischemic stroke induced by middle cerebral artery occlusion, whereas EPO had little effect. Similar to EPO, MK-X efficiently ameliorated mitochondrial dysfunction followed by neuronal death caused by glutamate-induced oxidative stress in cultured neurons. Consistent with this effect, MK-X significantly decreased caspase-3 cleavage and nuclear translocation of apoptosis-inducing factor induced by glutamate. MK-X completely mimicked the effect of EPO on multiple activation of JAK2 and its downstream PI3K/AKT and ERK1/2 signaling pathways, and this signaling process was involved in the neuroprotective effect of MK-X. Furthermore, MK-X and EPO induced similar changes in the gene expression patterns under glutamate-induced excitotoxicity. Interestingly, the most significant difference between MK-X and EPO was that MK-X better penetrated into the brain across the brain–blood barrier than did EPO. In conclusion, we suggest that MK-X might be used as a novel drug for protection from brain injury caused by ischemic stroke, which penetrates into the brain faster in comparison with EPO, even though MK-X and EPO have similar protective effects against excitotoxicity.

Abstract 361: Regulation of Activity of Apoptosis Inducing Factor by SUMO Conjugation

Apoptosis inducing factor (AIF), a mitochondrial oxidoreductase, is primarily involved in maintaining mitochondrial function. However, in response to apoptotic insults, AIF is released from mitochondria via cleavage and relocates to the nucleus, where it binds chromatin, subsequently causing its condensation and cell death. Thus, AIF plays an important role in both cell survival and death. The regulatory pathways/signaling events that govern the key step of AIF translocation and activity in cell death are not completely deciphered. SUMO conjugation, a highly conserved posttranslational modification, plays an important role in cardiac homeostasis. Herein, we report AIF as a novel SUMO substrate, and SENP5, a SUMO deconjugation enzyme, increased cardiomyocyte death once overexpressed in the mouse hearts, which coincided with an increase level of truncated AIF in cytosol. In line with the above findings, adenoviral mediated transduction of SENP5 wild type (wt), but not the C713L mutant, the latter of which lacks de-sumoylation activity, triggered cell death and AIF nuclear occupancy. Also, the SENP5-wt, but not the catalytically inactive mutant, de-sumoylated AIF. We further identified lysine residue 89 (K89) on AIF as one of the major SUMO attachment sites, and mutation of K89 to arginine (K89R) promoted AIF nuclear translocation in AC16, a cardiac cell line, and necrosis in L929 cells induced by TNFα/Z-vad-fmk. Our findings provide a novel mechanism by which SUMO conjugation is involved in mediating the function of AIF, which is important for cardiomyocyte survival and death.

Montelukast Induces Apoptosis-Inducing Factor-Mediated Cell Death of Lung Cancer Cells.

Developing novel chemo-prevention techniques and advancing treatment are key elements to beating lung cancer, the most common cause of cancer mortality worldwide. Our previous cohort study showed that cysteinyl leukotriene receptor antagonists, mainly montelukast, decreased the lung cancer risk in asthma patients. In the current study, we conducted in vivo and in vitro experiments to demonstrate the inhibiting effect of montelukast on lung cancer and to investigate the underlying mechanisms. Using Lewis lung carcinoma-bearing mice, we showed that feeding montelukast significantly delayed the tumor growth in mice (p < 0.0001). Montelukast inhibited cell proliferation and colony formation and induced the cell death of lung cancer cells. Further investigation showed the down-regulation of B-cell lymphoma 2 (Bcl-2), up-regulation of Bcl-2 homologous antagonist/killer (Bak), and nuclear translocation of apoptosis-inducing factor (AIF) in montelukast-treated lung cancer cells. Montelukast also markedly decreased the phosphorylation of several proteins, such as with no lysine 1 (WNK1), protein kinase B (Akt), extracellular signal-regulated kinase 1/2 (Erk1/2), MAPK/Erk kinase (MEK), and proline-rich Akt substrate of 40-kDa (PRAS40), which might contribute to cell death. In conclusion, montelukast induced lung cancer cell death via the nuclear translocation of AIF. This study confirmed the chemo-preventive effect of montelukast shown in our previous cohort study. The utility of montelukast in cancer prevention and treatment thus deserves further studies.

Synergistic induction of apoptosis by combination treatment with mesupron and auranofin in human breast cancer cells.

Urokinase-type plasminogen activator (uPA) has been validated as a predictive or prognostic biomarker protein, and mesupron is considered the first-in-class anticancer agent to inhibit uPA activity in human breast cancer. In the present study, we showed that the synergism between mesupron and auranofin, a thioredoxin reductase inhibitor, for inducing of apoptosis in MCF-7 human breast cancer cells. Our results demonstrated that mesupron and auranofin significantly lead to inhibition of the cancer cells proliferation; cell cycle arrest at the G1/S phase of the cell cycle, and apoptosis as indicated by caspase 3 activation, poly(ADP-ribose) polymerase cleavage, and annexin V staining. Isobologram analyses of MCF-7 cells showed a clear synergism between mesupron and auranofin. This combined treatment decreased the levels of mitochondrial anti-apoptotic factors, such as BCL-2, BCL-xL, and MCL-1 and caused nuclear translocation of apoptosis-inducing factor. Mitochondrial membrane potential (Δψ m ) was found to be strongly disrupted in combination-treated cells. In addition, combination treatment significantly enhanced the overproduction of reactive oxygen species, which was rescued by N-acetylcysteine treatment. The combination treatment suppressed phosphorylation of Akt, thus contributing to apoptosis. Taken together, our data suggest that the use of mesupron in combination with auranofin may be important in achieving high anticancer synergy.

Nutrient deprivation induces apoptosis of nucleus pulposus cells via activation of the BNIP3/AIF signalling pathway.

Nutrient deprivation (ND)-induced nucleus pulposus (NP) cell death serves an important role in intervertebral disc degeneration disease. However, the underlying mechanisms have yet to be thoroughly elucidated. The present study created a cell culture model under ND conditions to investigate the roles of the nutrient-sensitive protein B-cell lymphoma 2/adenovirus E1B 19 kDa-interacting protein (BNIP3) and the mitochondrial pro-death protein apoptosis-inducing factor (AIF) in the death pathway of NP cells. The present study demonstrated that cells subjected to ND for up to 72 h exhibited a time-dependent increase in cell death and decrease in mitochondrial membrane potential (Δψm), as compared with cells cultured under normal conditions. The results of western blotting demonstrated that BNIP3 expression was significantly upregulated in NP cells subjected to ND for 24 h, which coincided with AIF translocation to the cell nucleus and alterations in cell viability and Δψm. Furthermore, BNIP3 overexpression increased ND-induced NP cell death, whereas knockdown of BNIP3 or AIF abolished ND-induced NP cell death. In addition, BNIP3 overexpression increased AIF expression and BNIP3 knockdown decreased AIF expression in NP cells subjected to ND. In conclusion, ND induced NP cell death partially via activation of the BNIP3/AIF signalling pathway. These findings provide novel insights into the potential mechanisms underlying ND-induced death of NP cells during disc degeneration.

Oleuropein isolated from Fraxinus rhynchophylla inhibits glutamate-induced neuronal cell death by attenuating mitochondrial dysfunction

Glutamate-induced neurotoxicity is related to excessive oxidative stress accumulation and results in the increase of neuronal cell death. In addition, glutamate has been reported to lead to neurodegenerative diseases, including Parkinson's and Alzheimer's diseases.It is well known that Fraxinus rhynchophylla contains a significant level of oleuropein (Ole), which exerts various pharmacological effects. However, the mechanism of neuroprotective effects of Ole is still poorly defined. In this study, we aimed to investigate whether Ole prevents glutamate-induced toxicity in HT-22 hippocampal neuronal cells. The exposure of the glutamate treatment caused neuronal cell death through an alteration of Bax/Bcl-2 expression and translocation of mitochondrial apoptosis-inducing factor (AIF) to the cytoplasm of HT-22 cells. In addition, glutamate induced an increase in dephosphorylation of dynamin-related protein 1 (Drp1), mitochondrial fragmentation, and mitochondrial dysfunction. The pretreatment of Ole decreased Bax expression, increased Bcl-2 expression, and inhibited the translocation of mitochondrial AIF to the cytoplasm. Furthermore, Ole amended a glutamate-induced mitochondrial dynamic imbalance and reduced the number of cells with fragmented mitochondria, regulating the phosphorylation of Drp1 at amino acid residue serine 637. In conclusion, our results show that Ole has a preventive effect against glutamate-induced toxicity in HT-22 hippocampal neuronal cells. Therefore, these data imply that Ole may be an efficient approach for the treatment of neurodegenerative diseases.

Using connectivity map to identify natural lignan justicidin A as a NF-κB suppressor

Justicidin A (JA), a natural lignan, was isolated from the whole plant of Justicia procumbens, one of the most popular traditional Chinese medicines in China and Taiwan. Gene expression signatures of JA from human colorectal cancer HT-29 and hepatocellular carcinoma Hep 3B cells were used to query connectivity map. A NF-κB suppressor (15-delta prostaglandin J2) was predicted to display similar molecular action of JA. In JA-treated HT-29 cells, suppression of nuclear NF-κB expression and decrease of NF-κB DNA-binding activity were indeed observed. The classical pathway was involved in JA-induced inhibition of NF-κB, characterized by decreases in phosphorylation of AKT, IκB kinase (IKK)-β/IKK-α, and IκB-α. Furthermore, JA caused significant translocation of apoptosis-inducing factor and endonuclease G, caspase-independent apoptotic signaling molecules, from the mitochondrial to the nuclei. Our data suggest anti-inflammatory and cytotoxic mechanisms of JA, and using gene expression signatures to identify novel molecular actions of phytochemicals is an effective approach.

Drp1 mediates compression-induced programmed necrosis of rat nucleus pulposus cells by promoting mitochondrial translocation of p53 and nuclear translocation of AIF

Compression-induced programmed cell death of nucleus pulposus (NP) cells is an important contributor to intervertebral disc degeneration (IDD). Dynamin-related protein 1 (Drp1), a crucial mitochondrial fission protein, triggers programmed necrosis upon cellular injury. However, limited information is available about the role of Drp1 in compression-induced programmed necrosis of NP cells. In the present study, we found that compression resulted in upregulation and mitochondrial translocation of Drp1. Inhibition of Drp1 by siRNA or mitochondrial division inhibitor 1 (mdivi-1) effectively prevented the programmed necrosis of NP cells treated with compression. Furthermore, Drp1 promoted mitochondrial translocation of p53 and nuclear translocation of apoptosis-inducing factor (AIF) in compression-treated NP cells. Inhibition of p53 mitochondrial translocation by pifithrin-μ (PFT-μ) and silencing of AIF expression by siRNA significantly alleviated compression-induced NP cell programmed necrosis. These data indicates that Drp1 mediates compression-induced programmed necrosis of NP cells by promoting mitochondrial translocation of p53 and nuclear translocation of AIF.

Lanatoside C, a cardiac glycoside, acts through protein kinase C\u03b4 to cause apoptosis of human hepatocellular carcinoma cells

Recent studies have revealed that cardiac glycosides, such as digitalis and digoxin, have anticancer activity and may serve as lead compounds for the development of cancer treatments. The poor prognosis of hepatocellular carcinoma (HCC) patients reflects the development of resistance to current chemotherapeutic agents, highlighting the need for discovering new small-molecule therapeutics. Here, we found that lanatoside C, an anti-arrhythmic agent extracted from Digitalis lanata, inhibited the growth of HCC cells and dramatically decreased tumor volume as well as delayed tumor growth without obvious body weight loss. Moreover, lanatoside C triggered mitochondrial membrane potential (MMP) loss, activation of caspases and translocation of apoptosis-inducing factor (AIF) into the nucleus, which suggests that lanatoside C induced apoptosis through both caspase-dependent and -independent pathways. Furthermore, we discovered that lanatoside C activated protein kinase delta (PKCδ) via Thr505 phosphorylation and subsequent membrane translocation. Inhibition of PKCδ reversed lanatoside C-induced MMP loss and apoptosis, confirming that lanatoside C caused apoptosis through PKCδ activation. We also found that the AKT/mTOR pathway was negatively regulated by lanatoside C through PKCδ activation. In conclusion, we provide the first demonstration that the anticancer effects of lanatoside C are mainly attributable to PKCδ activation.

Visualization and Inhibition of Mitochondria-Nuclear Translocation of Apoptosis Inducing Factor by a Graphene Oxide-DNA Nanosensor.

High concentrations of oxidized low density lipoprotein (oxLDL) induce aberrant apoptosis of vascular smooth muscle cells (VSMCs) in atherosclerotic plaques. This apoptosis cannot be blocked completely by the inhibition of caspase, and it eventually potentiates plaque disruption and risk for cardiovascular disease. Given the important role of apoptosis inducing factor (AIF) in caspase-independent apoptosis, here we develop an AIF-targeting nanosensor by the assembly of graphene oxide (GO) nanosheets and dye-labeled DNA hybrid structures. This nanosensor selectively localizes in the cytosol of VSMCs, where it exhibits a "turn-off" fluorescence signal. Under oxLDL stimuli, the release of AIF from mitochondria into cytosol liberates the DNA hybrid structures from the surface of GO and results in a "turn-on" fluorescence signal. This nanosensor is shown to possess rapid response, high sensitivity, and selectivity for AIF that enables real-time imaging of AIF translocation in VSMCs. Using this novel nanosensor, a better assessment of the apoptotic level of VSMCs and a more accurate evaluation of the extent of atherosclerotic lesions can be obtained. More importantly, the abundant binding between DNA hybrid structures and AIF inhibits the translocation of AIF into the nucleus and subsequent apoptosis in VSMCs. This inhibition may help stabilize plaque and reduce the risk of heart attack and stroke.