BH3-interacting Domain Death Agonist Research Articles

Apoptosis induced by 9,11‑dehydroergosterol peroxide from GanodermaLucidum mycelium in human malignant melanoma cells is Mcl‑1 dependent.

9,11-Dehydroergosterol peroxide [9(11)-DHEP] is an important steroid from medicinal mushroom, which has been reported to exert antitumor activity in several tumor types. However, the role of 9(11)-DHEP toward the malignant melanoma cells has not been investigated. In the present study, the steroid from Ganoderma lucidum was purified on a submerged culture, and its antitumor mechanisms on A375 human malignant melanoma cells was investigated by MTT, flow cytometry and western blotting. The studies demonstrated that apoptotic mechanisms of the steroid were caspase-dependent and mediated via the mitochondrial pathway. The steroid did not cause significant changes in the expression levels of B-cell lymphoma 2 (Bcl-2) family proteins, Bcl-2-like protein 11, p53 upregulated modulator of apoptosis, Bcl-2-associated X protein, BH3 interacting-domain death agonist, Bcl-2-associated death promoter and Bcl-2, but it significantly downregulated induced myeloid leukemia cell differentiation protein Mcl-1 (Mcl-1) in melanoma cells, suggesting the key role of Mcl-1 in regulating apoptosis of melanoma cells induced by the steroid. These properties of 9(11)-DHEP advocate its usage as supplements in human malignant melanoma chemoprevention. The present study also suggests that mycelium of G. lucidum has a potential for producing bioactive substances and extracts with applications in medicine.

Increased A20-E3 ubiquitin ligase interactions in bid-deficient glia attenuate TLR3- and TLR4-induced inflammation

BackgroundChronic pro-inflammatory signaling propagates damage to neural tissue and affects the rate of disease progression. Increased activation of Toll-like receptors (TLRs), master regulators of the innate immune response, is implicated in the etiology of several neuropathologies including amyotrophic lateral sclerosis, Alzheimer’s disease, and Parkinson’s disease. Previously, we identified that the Bcl-2 family protein BH3-interacting domain death agonist (Bid) potentiates the TLR4-NF-κB pro-inflammatory response in glia, and specifically characterized an interaction between Bid and TNF receptor associated factor 6 (TRAF6) in microglia in response to TLR4 activation.MethodsWe assessed the activation of mitogen-activated protein kinase (MAPK) and interferon regulatory factor 3 (IRF3) inflammatory pathways in response to TLR3 and TLR4 agonists in wild-type (wt) and bid-deficient microglia and macrophages, using Western blot and qPCR, focusing on the response of the E3 ubiquitin ligases Pellino 1 (Peli1) and TRAF3 in the absence of microglial and astrocytic Bid. Additionally, by Western blot, we investigated the Bid-dependent turnover of Peli1 and TRAF3 in wt and bid−/− microglia using the proteasome inhibitor Bortezomib. Interactions between the de-ubiquitinating Smad6-A20 and the E3 ubiquitin ligases, TRAF3 and TRAF6, were determined by FLAG pull-down in TRAF6-FLAG or Smad6-FLAG overexpressing wt and bid-deficient mixed glia.ResultsWe elucidated a positive role of Bid in both TIR-domain-containing adapter-inducing interferon-β (TRIF)- and myeloid differentiation primary response 88 (MyD88)-dependent pathways downstream of TLR4, concurrently implicating TLR3-induced inflammation. We identified that Peli1 mRNA levels were significantly reduced in PolyI:C- and lipopolysaccharide (LPS)-stimulated bid-deficient microglia, suggesting disturbed IRF3 activation. Differential regulation of TRAF3 and Peli1, both essential E3 ubiquitin ligases facilitating TRIF-dependent signaling, was observed between wt and bid−/− microglia and astrocytes. bid deficiency resulted in increased A20-E3 ubiquitin ligase protein interactions in glia, specifically A20-TRAF6 and A20-TRAF3, implicating enhanced de-ubiquitination as the mechanism of action by which E3 ligase activity is perturbed. Furthermore, Smad6-facilitated recruitment of the de-ubiquitinase A20 to E3-ligases occurred in a bid-dependent manner.ConclusionsThis study demonstrates that Bid promotes E3 ubiquitin ligase-mediated signaling downstream of TLR3 and TLR4 and provides further evidence for the potential of Bid inhibition as a therapeutic for the attenuation of the robust pro-inflammatory response culminating in TLR activation.

Hypertonicity-enforced BCL-2 addiction unleashes the cytotoxic potential of death receptors

Attempts to exploit the cytotoxic activity of death receptors (DR) for treating cancer have thus far been disappointing. DR activation in most malignant cells fails to trigger cell death and may even promote tumor growth by activating cell death-independent DR-associated signaling pathways. Overcoming apoptosis resistance is consequently a prerequisite for successful clinical exploitation of DR stimulation. Here we show that hyperosmotic stress in the tumor microenvironment unleashes the deadly potential of DRs by enforcing BCL-2 addiction of cancer cells. Hypertonicity robustly enhanced cytotoxicity of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and other DR ligands in various cancer entities. Initial events in TRAIL DR signaling remained unaffected, but hypertonic conditions unlocked activation of the mitochondrial death pathway and thus amplified the apoptotic signal. Mechanistically, we demonstrate that hyperosmotic stress imposed a BCL-2-addiction on cancer cells to safeguard the integrity of the outer mitochondrial membrane (OMM), essentially exhausting the protective capacity of BCL-2-like pro-survival proteins. Deprivation of these mitochondrial safeguards licensed DR-generated truncated BH3-interacting domain death agonist (tBID) to activate BCL-2-associated X protein (BAX) and initiated mitochondrial outer membrane permeabilization (MOMP). Our work highlights that hyperosmotic stress in the tumor environment primes mitochondria for death and lowers the threshold for DR-induced apoptosis. Beyond TRAIL-based therapies, our findings could help to strengthen the efficacy of other apoptosis-inducing cancer treatment regimens.

Ameliorative effect of nicergoline on cognitive function through the PI3K/AKT signaling pathway in mouse models of Alzheimer's disease.

Alzheimer's disease is one of the most common age‑associated diseases that frequently leads to memory disorders, cognitive decline and dementia. Evidence suggests that nicergoline serves an important role in the apoptosis of hippocampal cells, memory recovery, cognitive function and neuronal survival. However, the signaling pathway affected by nicergoline treatment remains to be elucidated. The purpose of the present study was to investigate the role of nicergoline in the cognitive competence of a mouse model of Alzheimer's disease. The apoptosis rates of hippocampal cells were studied in mice with Alzheimer's disease treated with nicergoline compared with the negative control. Apoptosis‑associated gene expression levels in hippocampal cells, and hippocampus area, were analyzed in the experimental mice. Visual attention and inhibitory control were assessed and neural counting was performed in brain regions of interest. The phosphatidylinositol 3‑kinase (PI3K)/RAC‑α serine/threonine‑protein kinase (AKT) signaling pathway was additionally analyzed in hippocampal cells following treatment with nicergoline. The results of the present study demonstrated that nicergoline ameliorated apoptosis in hippocampal cells and hippocampus tissue in 3xTg‑AD mice with Alzheimer's disease. The data indicated that apoptosis‑associated genes, including caspase‑3, BCL2 associated X, BH3 interacting domain death agonist and caspase‑9, were downregulated in hippocampal cells isolated from nicergoline-treated experimental mice. In addition, the expression levels of inflammatory factors, in addition to oxidative stress, were decreased in hippocampal cells treated with nicergoline. Additionally, amyloid precursor protein accumulation was cleared in the hippocampal area in nicergoline‑treated mice. Nicergoline inhibited neuronal loss and prevented cognitive impairment through the restoration of learning/memory ability. It was additionally demonstrated in the present study that nicergoline improved motor attention impairment and cognitive competence in hippocampal cells by acting on the PI3K/AKT signaling pathway. Therefore, memory recovery, cognitive function and neuronal survival were repaired by nicergoline via inhibition of the PI3K/AKT signaling pathway, suggesting that nicergoline may be an efficient drug for the clinical treatment of patients with Alzheimer's disease.

Loss of BID Delays FASL-Induced Cell Death of Mouse Neutrophils and Aggravates DSS-Induced Weight Loss.

Neutrophils are key players in the early defense against invading pathogens. Due to their potent effector functions, programmed cell death of activated neutrophils has to be tightly controlled; however, its underlying mechanisms remain unclear. Fas ligand (FASL/CD95L) has been shown to induce neutrophil apoptosis, which is accelerated by the processing of the BH3-only protein BH3 interacting domain death agonist (BID) to trigger mitochondrial apoptotic events, and been attributed a regulatory role during viral and bacterial infections. Here, we show that, in accordance with previous works, mouse neutrophils underwent caspase-dependent apoptosis in response to FASL, and that this cell death was significantly delayed upon loss of BID. However, pan-caspase inhibition failed to protect mouse neutrophils from FASL-induced apoptosis and caused a switch to RIPK3-dependent necroptotic cell death. Intriguingly, such a switch was less evident in the absence of BID, particularly under inflammatory conditions. Delayed neutrophil apoptosis has been implicated in several auto-inflammatory diseases, including inflammatory bowel disease. We show that neutrophil and macrophage driven acute dextran sulfate sodium (DSS) induced colitis was slightly more aggravated in BID-deficient mice, based on significantly increased weight loss compared to wild-type controls. Taken together, our data support a central role for FASL > FAS and BID in mouse neutrophil cell death and further underline the anti-inflammatory role of BID.

Gambogic acid sensitizes breast cancer cells to TRAIL-induced apoptosis by promoting the crosstalk of extrinsic and intrinsic apoptotic signalings

Due to the ability of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) to induce cancer cell apoptosis selectively, TRAIL has attracted significant interest in the treatment of cancer. However, although TRAIL triggers apoptosis in a broad range of cancer cells, most primary cancers are often intrinsically TRAIL-resistant, or can acquire resistance after TRAIL treatment, evocating new strategies to overcome TRAIL resistance. Gambogic acid (GA), an active constituent of Garcinia Hanburyi (Teng Huang in Chinese), has been applied for thousands of years for medicinal uses, however, the potential effect of GA in combating cancer resistance remains poorly investigated. In this study, we found that GA could increase the sensitivity of breast cancer cells to TRAIL and enhance TRAIL-induced apoptosis. GA cooperated with TRAIL to decrease the levels of anti-apoptotic proteins and activate Bid (BH3 interacting-domain death agonist) to promote the crosstalk of extrinsic and intrinsic apoptotic signaling, rather than increasing the expression of TRAIL receptors DR4 and DR5. These findings may open a new window in the treatment of breast cancer using TRAIL in combination with GA.

Minocycline impedes mitochondrial-dependent cell death and stabilizes expression of hypoxia inducible factor-1α in spinal cord injury.

IntroductionOne of the crucial mechanisms following spinal cord injury is mitochondria-associated cell death. Minocycline, an anti-inflammatory drug, is well known to impede mitochondrial cell death. However, there has been no study on the effect of minocycline linking Fas cell surface death receptor (FAS)-mediated cell death and hypoxia inducible factor (HIF-1α), the targets involved in mitochondrial cell death.Material and methodsMale Sprague Dawley rats (N = 15, divided into three groups) were subjected to traumatic spinal cord injury and were injected with minocycline (n = 5) (90 mg/kg and later a 45 mg/kg dose twice a day (every 12 h)). Injection with sterile PBS in injured animals served as the vehicle (n = 5) and another group comprised healthy animals (n = 5). TUNEL assay was used to quantify cell death. The release of Smac/Diablo, cytochrome-c (cyt-c), HIF-1α, FAS ligand (FASL) and tumour necrosis factor-α (TNF-α) was measured using ELISA. Expression of HIF-1α, FASL and other cell death associated factors was quantified at the mRNA and protein level and confirmed with immunohistochemistry.ResultsThere was a marked reduction in the HIF-1α and FASL expression levels in the minocycline-treated group compared to the vehicle. The reduction of HIF-1α and FASL was associated with other factors linked to cell death (Smac/Diablo, cyt-c, TNF-α, p53, caspase-8 and BH3 interacting domain death agonist (BID)) (p < 0.5; *p < 0.05 vs. vehicle group, **p < 0.01 vs. vehicle group).ConclusionsThe present study focuses on the investigation of minocycline in inhibiting mitochondria-associated cell death by modulating FASL and HIF-1α expression, which are seemingly interlinked mechanisms contributing to cell death.

Paeoniflorin induces G2/M cell cycle arrest and caspase-dependent apoptosis through the upregulation of Bcl-2 X-associated protein and downregulation of B-cell lymphoma 2 in human osteosarcoma cells.

Paeoniflorin (PF), extracted from the peony root, has been proved to possess antineoplastic activity in different cancer cell lines. However, it remains unclear whether PF has an antineoplastic effect against osteosarcoma cells. The present study investigated the effects and the specific mechanism of PF on various human osteosarcoma cell lines. Using the multiple methods to detect the activity of PF on HOS and Saos-2 human osteosarcoma cell lines, including an MTS assay, flow cytometry, transmission electron microscopy and western blotting, it was demonstrated that PF induces inhibition of proliferation, G2/M phase cell cycle arrest and apoptosis in the osteosarcoma cell lines in vitro, and activation of cleaved-caspase-3 and cleaved-poly (ADPribose) polymerase in a dose-dependent manner. Furthermore, the pro-apoptotic factors Bcl-2 X-associated protein and BH3 interacting domain death agonist were uregulated, while the anti-apoptotic factors B-cell lymphoma 2 (Bcl-2) and Bcl-2-extra large were downregulated. In conclusion, these results demonstrated that PF has a promising therapeutic potential in for osteosarcoma.

Eudesmin attenuates Helicobacter pylori-induced epithelial autophagy and apoptosis and leads to eradication of H. pylori infection.

Eudesmin has been proven to possess anti-inflammatory effects. In the present study, the effects of eudesmin on Helicobacter pylori (H. pylori)-mediated autophagy, apoptosis, immune response and inflammation were determined in human gastric adenocarcinoma (AGS) cells in vitro and in C57BL/6 mice in vivo. Detection of the production of interleukin (IL)-8, IL-1β and immunoglobulin M (IgM) was performed using ELISA. Identification of the activation of apoptosis-associated caspase-3, −8 and −9 proteins, Bcl-2-associated X protein (Bax) and BH3 interacting domain death agonist (Bid) protein, was determined through western blot analysis. Autophagy microtubule-associated protein 1A/1B-light chain 3, isoform B (LC-3B) expression was measured using immunostaining. The results of the present study demonstrated that eudesmin inhibited the growth of H. pylori, with increased inhibition activity against antibiotic resistant strains compared with the reference strain. In addition, H. pylori-induced IL-8 secretion, LC-3B expression and apoptosis-associated protein (caspase-3, −8 and −9, Bax and Bid) activation in AGS cells was suppressed by eudesmin. Furthermore, eudesmin suppressed IL-1β and IgM production in H. pylori-infected C57BL/6 mice in vivo. In conclusion, eudesmin may be developed as a promising therapeutic agent to prevent and/or treat H. pylori-associated gastric inflammation.

Novel Combination of Arsenic Trioxide (As2O3) Plus Resveratrol in Inducing Programmed Cell Death of Human Neuroblastoma SK-N-SH Cells.

Arsenic trioxide (As2O3), known as pi-shuang and the most toxic compound in traditional Chinese medicine, has been used as an antitumor agent for thousands of years. Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a natural phenol that has significant anti-bacterial, anti-fungaI and antiaging activities. Our study aimed to examine the combined anticancer effects of As2O3 and resveratrol against human neuroblastoma SK-N-SH cells, and elucidate the underlying intracellular signaling. SK-N-SH cells were treated with an extremely low-dose (2-4 μM) of As2O3 alone or combined with 75 μg/ml resveratrol for further comparisons. Cell viability, apoptotic signaling as well as synergistic cytotoxic effects were estimated using the MTT assay, microscopy observation, flow cytometric analysis for loss of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS), and typical quantitative western blotting analysis. Student's t-test, and one- and two-way analysis of variance (ANOVA) were used for examination of significant differences. The combined treatment was more effective than single treatment of As2O3 or resveratrol alone in suppressing cell viability, which correlated with the elevation of ROS levels. The intracellular mechanisms of cytotoxicity of As2O3 plus resveratrol were revealed as ROS accumulation and relative decrease of MMP, leading to activation of caspase-3 and -9, but not of caspase-1, -7 and-8. Combination treatment reduced the expression of B-cell lymphoma 2 (BCL2), BH3 interacting domain death agonist (BID), and BCL-x/L. Combined treatment at extremely low concentration of two agents from natural products, As2O3 and resveratrol, has high potential as a cocktail of anticancer drugs for neuroblastoma.

Escitalopram oxalate inhibits proliferation and migration and induces apoptosis in non-small cell lung cancer cells.

Population-based cohort studies have revealed that neuroleptic medications are associated with a reduced cancer risk. Recent studies have demonstrated that selective serotonin reuptake inhibitors (SSRIs) have an antiproliferative or cytotoxic effect on certain cancer types. Known as a superior SSRI, escitalopram oxalate exhibits favorable tolerability with generally mild and temporary adverse events. The present study aimed to examine the effects of escitalopram oxalate on non-small cell lung cancer (NSCLC) cells. The experimental results revealed that escitalopram oxalate significantly inhibited the proliferation and invasion of A549, and H460 cells compared with BEAS-2B cells. Additionally, escitalopram oxalate significantly increased the sub-G1 population and caspase-3 activity of A549, and H460 cells. Furthermore, escitalopram oxalate significantly induced mitochondria-dependent apoptotic signaling cascades in A549 and H460 cells, which included increases in the protein expression levels of apoptosis regulator Bax, truncated BH3-interacting domain death agonist, cytochrome c, apoptotic protease-activating factor 1, and cleaved caspase-9. These findings suggest that escitalopram oxalate could serve a therapeutic agent for the treatment of NSCLC due to its antiproliferative and apoptotic effects.

112 Identification of Solute Carrier Family 5 Member 9 (SLC5A9) as Biomarker of In Vivo and In Vitro Oocyte Developmental Competence

The transcriptome associated with the ability of oocytes to support embryogenesis and development remains unclear. The aim of this study was to compare the mRNA expression pattern between oocytes collected from fertile heifers (H) and repeat breeders (RB) using RNAseq. Oocytes were collected by ovum pick-up from Italian Simmental (IS) and Holstein Friesian (HF) heifers that became pregnant at the following oestrus, and from cows of the same breeds classified as RB after they failed to become pregnant for a minimum of 3 consecutive AI. Oocytes from each animal (5 for HF_RB, 3 each for all other groups) were pooled and sequenced using Illumina HiSEqn 2000 (Illumina Inc., San Diego, CA, USA). Principal component analysis showed that HF oocytes clustered away from those of IS, irrespective of the fertility status. However, within breeds, H and RB always clustered apart from each other. This suggests that differences in the transcriptional blueprint between breeds was more prominent than differences related to fertility. However, these were still strong when considering breeds separately, therefore we treated breeds as independent groups and performed differential expression analysis on H v. RB in IS and HF. DESEqn 2 identified 135 differentially expressed genes when we compared H and RB in IS and 2124 in HF. This difference in number of DE genes between the 2 breeds reflects the fact that the variance between H and RB transcriptional patterns in HF is larger than in IS. We looked for genes that were differentially and consistently expressed between H and RB in both breeds and identified 10 genes: histone H2B, exo/endonuclease G, solute carrier family 5 member 9, BH3 interacting domain death agonist, TBK1 binding protein 1, purine-rich element binding protein A, tRNA-yW synthesising protein 1 homologue, Jun oncogene, FRA10AC1 homologue, and SKI oncogene. To validate their potential use as biomarkers, we performed a preliminary in vitro validation by RT-qPCR. As described in the literature, oocytes collected from ovaries with more than 10 follicles of 2 to 6 mm were classified as high quality, while those collected from ovaries with fewer than 10 follicles were classified as low quality (Gandolfi et al. 1997 Theriogenology 48, 1153-1160). Gene expression analysis confirmed SLC5A9, a membrane-bound protein involved in sodium-dependent transport of glucose, as a potential biomarker to discriminate between a competent and incompetent oocyte. Altogether, our data provide a detailed overview of the mRNA pattern defining the differences between a competent and incompetent oocyte in vivo, and identified a new potential marker able to differentiate fertile and infertile oocyte both in vivo and in vitro. Study supported by PRIN 08–09, EU-Quantomics no. 222664 and Carraresi Foundation.

Cell death and restoration of TRAIL-sensitivity by ciglitazone in resistant cervical cancer cells.

Known activators of the Peroxisome Proliferator-Activated Receptor γ (PPARγ), thiazolidinediones (TZD) induce apoptosis in a variety of cancer cells through dependent and/or independent mechanisms of the receptor. We tested a panel of TZD (Rosiglitazone, Pioglitazone, Ciglitazone) to shed light on their potential therapeutic effects on three cervical cancer cell lines (HeLa, Ca Ski, C-33 A). In these cells, only ciglitazone triggered apoptosis through PPARγ-independent mechanisms and in particular via both extrinsic and intrinsic pathways in Ca Ski cells containing Human PapillomaVirus (HPV) type 16. It also inhibits cervical cancer xenograft development in nude mice. Ciglitazone kills cervical cancer cells by activating death receptor signalling pathway, caspase cascade and BH3 interacting-domain death agonist (Bid) cleavage through the up-regulation of Death Receptor 4 (DR4)/DR5 and soluble and membrane-bound TNF related apoptosis inducing ligand (TRAIL). Importantly, the drug let TRAIL-resistant Ca Ski cells to respond to TRAIL through the downregulation of cellular FLICE-Like Inhibitory Protein (c-FLIP) level. For the first time, we revealed that ciglitazone is able to decrease E6 viral oncoprotein expression known to block TRAIL pathway and this was associated with cell death. Our results highlight the capacity of ciglitazone to restore TRAIL sensitivity and to prevent E6 blocking action to induce apoptosis in cervical cancer cells.

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.

Hesperidin induces apoptosis and G0/G1 arrest in human non-small cell lung cancer A549 cells.

Lung cancer has high incidence and mortality rates worldwide. In the present study, the mechanisms by which hesperidin decreases the viability and induces the apoptosis of human non-small cell lung cancer(NSCLC) A549 cells were investigated. Initially, MTT and flow cytometric assays were performed to evaluate the effects of hesperidin on the viability and apoptosis of A549 cells and human normal lung epithelial BEAS-2B cells. The results revealed that hesperidin has no negative effects on the human normal lung epithelial BEAS-2B cells and the viability of cells treated with various concentrations of hesperidin was inhibited in a time- and dose-dependent manner compared with the control groups. Subsequently, the expression levels of proteins involved in the mitochondria-associated apoptotic pathway were studied by western blot analysis. Hesperidin was identified to induce A549 cell apoptosis by downregulating the levels of B-cell lymphoma-2(Bcl-2) and Bcl extra large protein and simultaneously upregulating the levels of Bcl-2-associated Xprotein, BH3 interacting-domain death agonist(Bid), tBid, cleaved caspase-9, cleaved caspase-3 and cleaved poly(adenosine diphosphate ribose)polymerase. The effect of hesperidin on the cell cycle was assessed by flow cytometry. Hesperidin was observed to cause G0/G1 arrest of A549 cells by decreasing the expression of cyclinD1 and increasing the expression of p21 and p53. In summary, it was demonstrated that hesperidin induced apoptosis through the mitochondrial apoptotic pathway and induced G0/G1 arrest in human NSCLC A549 cells. Therefore, hesperidin may be developed as a potential therapeutic drug for the treatment of NSCLC.

Signal-Regulated Protein Kinases/Protein Kinase B-p53-BH3-Interacting Domain Death Agonist Pathway Regulates Gingipain-Induced Apoptosis in Osteoblasts.

Gingipains are cysteine proteases produced by Porphyromonas gingivalis, the predominant pathogen in chronic periodontitis. The present study aims to examine the role of gingipains in promoting apoptosis in osteoblasts. Human calvarial osteoblasts and osteoblast MC3T3-E1 cells were treated with 8.348 U/L gingipains. Flow cytometry analysis and terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling staining were used to detect cell apoptosis. Protein expression was examined by Western blotting, and gene expression was detected by real-time polymerase chain reaction. Small interfering (si)RNA transfection was used to knock down BH3-interacting domain death agonist (Bid) expression. Treatment with 8.348 U/L gingipains from 4 to 72 hours increased apoptosis, accompanied by elevated cleaved caspase-3 levels. Notably, gingipain-induced apoptosis was associated with increase of Bid and its truncated form, tBid, as well as p53. Transfection with Bid siRNA resulted in suppression of gingipain-induced apoptosis. The p53 inhibitor, Pifithrin-α, blocked the gingipain-induced Bid. The ability of gingipains to stimulate p53 and Bid expression was mimicked by PD-0325901 and MK-2206, the specific extracellular signal-regulated protein kinases (ERK) and protein kinase B (PKB) inhibitors, respectively. Furthermore, treatment with gingipains reduced phospho-ERK and phospho-PKB levels, an effect correlated to gingipain-induced increase in p53 and tBid expression. The present findings suggest that Bid plays an essential role in gingipain-induced osteoblast apoptosis, which is dependent on inhibition of ERK and PKB phosphorylation, followed by the activation of p53.

Fas cell surface death receptor controls hepatic lipid metabolism by regulating mitochondrial function

Nonalcoholic fatty liver disease is one of the most prevalent metabolic disorders and it tightly associates with obesity, type 2 diabetes, and cardiovascular disease. Reduced mitochondrial lipid oxidation contributes to hepatic fatty acid accumulation. Here, we show that the Fas cell surface death receptor (Fas/CD95/Apo-1) regulates hepatic mitochondrial metabolism. Hepatic Fas overexpression in chow-fed mice compromises fatty acid oxidation, mitochondrial respiration, and the abundance of mitochondrial respiratory complexes promoting hepatic lipid accumulation and insulin resistance. In line, hepatocyte-specific ablation of Fas improves mitochondrial function and ameliorates high-fat-diet-induced hepatic steatosis, glucose tolerance, and insulin resistance. Mechanistically, Fas impairs fatty acid oxidation via the BH3 interacting-domain death agonist (BID). Mice with genetic or pharmacological inhibition of BID are protected from Fas-mediated impairment of mitochondrial oxidation and hepatic steatosis. We suggest Fas as a potential novel therapeutic target to treat obesity-associated fatty liver and insulin resistance.

Bid-deficient fish delay grass carp reovirus (GCRV) replication and attenuate GCRV-triggered apoptosis.

Bid, BH3-interacting domain death agonist, is a pro-apoptotic BH3-only member of Bcl-2 family, playing an important role in apoptosis. In the study, Bid genes from grass carp (Ctenopharyngodon idellus) and rare minnow (Gobiocypris rarus), named CiBid and GrBid, were cloned and analyzed. Bid was constitutively expressed in all examined tissues of grass carp, but the expression level varied in different tissues. Following grass carp reovirus (GCRV) stimulation in vivo, Bid and apoptosis related genes Caspase-9 and Caspase-3 was up-regulated significantly at the late stage of infection. Moreover, we generated a Bid-deficient rare minnow (Bid-/-) to investigate the possible role of Bid in GCRV-triggered apoptosis. We found that the survival time of Bid-/- rare minnow after GCRV infection was extended when compared with wild-type fish, the relative copy number of GCRV in Bid-/- rare minnow was lower than that in wild-type fish, and the expression level of Caspase-9 and Caspase-3 in Bid-/- rare minnow were significantly lower than that in the wild-type fish. Collectively, the current data revealed the important role of Bid during virus-induced apoptosis in teleost fish. Our study would provide new insight into understanding the GCRV induced apoptosis and may provide a target gene for virus-resistant breeding in grass carp.

Hyperthermia and radiation reduce the toxic side-effects of bufadienolides for cancer therapy.

Bufadienolides are constituents of the traditional Chinese medicine Chan Su and are found in toad venom. Cardiovascular side-effects are one of the limiting factors towards developing bufadienolides as chemotherapeutic agents. Thus, in the present study, low doses of bufalin and cinobufotalin, prominent members of the bufadienolides, were investigated for their cytotoxic activity in combination with hyperthermia (HT) or radiation (Rad) therapy. In addition, the underlying mechanism involved was investigated. A DNA fragmentation assay, viability assay and microscopic observation were primarily used to assess the effect of low doses of the two drugs in human lymphoma U937 cells. Furthermore, the effects of these drugs on the mitochondrial membrane potential (MMP) and apoptotic-associated protein activation were investigated. HT/bufadienolide- and RT/bufadienolide-treated samples significantly increased the DNA fragmentation percentile and decreased the MMP, as well as increasing the apoptotic features observed microscopically within a relatively short time (6 h) after treatment. The two combinations affected the expression of important apoptotic markers, including caspase-3 and BH3 interacting domain death agonist. The findings of the current study confirm the additive effect of HT with this group of drugs, directing a novel therapeutic avenue for the clinical use of bufadienolides at lower doses with more restrained cardio toxic side-effects.

Knockdown of ZFPL1 results in increased autophagy and autophagy‑related cell death in NCI‑N87 and BGC‑823 human gastric carcinoma cell lines.

Macroautophagy, which will hereafter be referred to as autophagy, is an evolutionarily conserved process, during which cells recycle and remove damaged organelles and proteins in response to cellular stress. However, the mechanisms underlying the regulation of autophagy remain to be fully elucidated. The present study demonstrated that knockdown of zinc finger protein like1 (ZFPL1) induces autophagy and increases autophagic cell death in NCI‑N87 and BGC‑823 human gastric carcinoma cell lines. To examine the role of ZFPL1 in gastric carcinoma cells, ZFPL1 expression was downregulated by lentiviral infection. Zinc finger domain‑FLAG was used to compete with ZFPL1 for golginA2/GM130 binding. Autophagy was analyzed by red fluorescent protein‑microtubule‑associated protein 1A/1B‑light chain3 (LC3) puncta, LC3I to LC3II conversion, and p62 expression. The results demonstrated that knockdown of ZFPL1 was able to significantly increase cell death rate. However, ZFPL1 knockdown exerted almost no effect on the expression of apoptosis‑associated markers, including Bcell lymphoma2 (Bcl‑2), Bcl‑x, Bcl‑2‑associated X protein, BH3 interacting domain death agonist, p53, and the classical caspase family members, caspase‑3, caspase‑8 and caspase‑9. An endogenous ZFPL1‑GM130 association was identified in NCI‑N87 cells and BGC‑823 cells by co‑immunoprecipitation. Furthermore, cell death was restricted following treatment of ZFPL1 knockdown cells with an autophagy inhibitor. Therefore, knockdown of ZFPL1 expression may induce cell death via autophagy, rather than apoptosis. These results suggest that ZFPL1 may serve an important role in regulating autophagy in NCI‑N87 and BGC‑823 cells.