Interacting Domain Death Agonist Research Articles

The BH3-only protein BID impairs the p38-mediated stress response and promotes hepatocarcinogenesis during chronic liver injury in mice.

Apoptosis is critical for maintaining tissue homeostasis, and apoptosis evasion is considered as a hallmark of cancer. However, increasing evidence also suggests that proapoptotic molecules can contribute to the development of cancer, including liver cancer. The aim of this study was to further clarify the role of the proapoptotic B-cell lymphoma 2 homology domain 3 (BH3)-only protein BH3 interacting-domain death agonist (BID) for chronic liver injury (CLI) and hepatocarcinogenesis (HCG). Loss of BID significantly delayed tumor development in two mouse models of Fah-mediated and HBsTg-driven HCG, suggesting a tumor-promoting effect of BID. Liver injury as well as basal and mitogen-stimulated hepatocyte proliferation were not modulated by BID. Moreover, there was no in vivo or in vitro evidence that BID was involved in DNA damage response in hepatocytes and hepatoma cells. Our data revealed that CLI was associated with strong activation of oxidative stress (OS) response and that BID impaired full activation of p38 after OS. We provide evidence that the tumor-promoting function of BID in CLI is not related to enhanced proliferation or an impaired DNA damage response. In contrast, BID suppresses p38 activity and facilitates malignant transformation of hepatocytes.

Nortriptyline induces mitochondria and death receptor-mediated apoptosis in bladder cancer cells and inhibits bladder tumor growth in vivo

Nortriptyline (NTP), an antidepressant, has antitumor effects on some human cancer cells, but its effect on human bladder cancer cells is not known. In this study, we used a cell viability assay to demonstrate that NTP is cytotoxic to human TCCSUP and mouse MBT-2 bladder cancer cells in a concentration and time-dependent manner. We also performed cell cycle analysis, annexin V and mitochondrial membrane potential assays, and Western blot analysis to show that NTP inhibits cell growth in these cells by inducing both mitochondria-mediated and death receptor-mediated apoptosis. Specifically, NTP increases the expression of Fas, FasL, FADD, Bax, Bak, and cleaved forms of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. In addition, NTP decreases the expression of Bcl-2, Bcl-xL, BH3 interacting domain death agonist, X-linked inhibitor of apoptosis protein, and survivin. Furthermore, NTP-induced apoptosis is associated with reactive oxygen species (ROS) production, which can be reduced by antioxidants, such as N-acetyl-L-cysteine. Finally, we showed that NTP suppresses tumor growth in mice inoculated with MBT-2 cells. Collectively, our results suggest that NTP induces both intrinsic and extrinsic apoptosis in human and mouse bladder cancer cells and that it may be a clinically useful chemotherapeutic agent for bladder cancer in humans.

Quercetin-3-O-(2″-galloyl)-α-L-rhamnopyranoside attenuates cholesterol oxidation product-induced apoptosis by suppressing NF-κB-mediated cell death process in differentiated PC12 cells.

Cholesterol oxidation products are suggested to be involved in neuronal cell degeneration. We examined the preventive effect of quercetin-3-O-(2″-galloyl)-α-L-rhamnopyranoside (QGR), a quercetin derivative, on the cholesterol oxidation product-induced neuronal cell death using differentiated PC12 cells in relation to nuclear factor (NF)-κB-mediated apoptotic process. 7-Ketocholesterol and 25-hydroxycholesterol induced a decrease in the levels of BH3 interacting-domain death agonist (Bid) and B cell lymphoma 2 (Bcl-2), increase in the levels of Bcl-2-associated X protein (Bax) and p53, loss of the mitochondrial transmembrane potential, cytochrome c release, activation of caspases, and cleavage of poly(ADP-ribose) polymerase 1 (PARP-1). 7-Ketocholesterol induced increase in cytosolic and nuclear NF-κB p65, nuclear phospho-NF-κB p65, cytosolic NF-κB p50, and cytosolic phospho-IκB-α levels. The addition of QGR, N-acetyl cysteine, or Bay 11-7085 attenuated the cholesterol oxidation product-induced changes in the apoptosis-related protein levels, activation of NF-κB, formation of reactive oxygen species, depletion of glutathione (GSH), nuclear damage, and cell death. The results show that QGR may attenuate the cholesterol oxidation product-induced apoptosis in PC12 cells by suppressing the activation of the mitochondrial pathway and the caspase-8- and Bid-dependent pathways that is mediated by NF-κB activation. The preventive effect appears to be associated with the inhibitory effect on the formation of reactive oxygen species and depletion of GSH.

Circadian gene hClock enhances proliferation and inhibits apoptosis of human colorectal carcinoma cells invitro and invivo.

Colorectal carcinoma (CRC) is one of the most prevalent types of malignancy-associated mortality worldwide. Previous studies have demonstrated that amplification and overexpression of the human circadian locomotor output cycles kaput gene (hClock) was closely associated with a high risk for CRC as well as poor prognosis in CRC patients. However, the underlying molecular mechanisms of CRC remain to be fully elucidated. In the present study, hClock was exogenously overexpressed in the CRC cell line SW480 via infection of a lentivirus vector expressing hClock; in addition, a lentivirus vector-based RNA interference approach, using short hairpin RNA, was performed in order to knockdown hClock in SW620 cells. The results showed that upregulation of hClock promoted proliferation and inhibited apoptosis in SW480 cells in vitro and in vivo, while downregulation of hClock inhibited SW620 cell proliferation and accelerated apoptosis in vitro. Upregulation of hClock enhanced the activity of the anti-apoptotic gene phosphorpylated (p-) AKT and inhibited the expression of the pro-apoptotic gene B cell lymphoma-2 (Bcl-2)-associated X protein and Bcl-2 homology 3 interacting domain death agonist. Furthermore, targeted inhibition of hClock activity reduced p-AKT expression. In conclusion, the results of the present study suggested that the circadian gene hClock promoted CRC progression and inhibit tumor cell apoptosis in vitro and in vivo, while silencing hClock was able to reverse this effect.

The effects of grape seed on apoptosis-related gene expression and oxidative stress in streptozotocin-induced diabetic rats

Background: Diabetic nephropathy is the most common cause of end-stage renal disease. Emerging evidences indicate that many mechanistic pathways including apoptosis play an important role in the pathogenesis and progression of macrovascular and microvascular complications of diabetes mellitus. The aim of the present study is to show the effects of grape seed extract (GSE) on oxidative stress and apoptosis in the kidney of streptozotocin-induced diabetic rats. Materials and methods: The study included control group, diabetic group without treatment and diabetic group treated with GSE (n = 7) group. GSE was given orally (100 mg/kg/day) for six weeks. Following parameters were evaluated; oxidative stress index, caspase 1, IL1-alpha, caspase 2, IL1-beta, BCL2-associated agonist of cell death (BAD), X-linked inhibitor of apoptosis (XIAP), DNA fragmentation factor, alpha subunit and beta bubunit (DFFA, DFFB), BH3 interacting domain death agonist (BID), caspase 6, Bcl2-like 1 (BCL-XL), caspase 8, tumor necrosis factor receptor superfamily, member 1 b (TNFRSF1B) and IAP-binding mitochondrial protein (DIABLO). Results: Oxidative stress index levels were significantly increased in the kidney of diabetic group without treatment compared to control group, and decreased in diabetic + GSE group compared to diabetic group without treatment. In the kidney of diabetic group without treatment, caspase 1, IL-1 alpha, BAD, DFFA, DFFB and caspase-6 gene expressions were significantly higher compared to control group. In diabetic + GSE group caspase 1, caspase 2, XIAP, DFFA, BID, BCL-XL and TNFRSF1B genes were significantly decreased compared to control group. Conclusions: Grape seed reduces oxidative stress and apoptosis gene expression suggesting the protective effect on diabetic nephropathy.

Heptaphylline induces apoptosis in human colon adenocarcinoma cells through bid and Akt/NF-κB (p65) pathways.

Heptaphylline derivatives are carbazoles in Clausena harmandiana, a medicinal plant that is utilized for headache, stomach ache, and other treatments of illness. The present study examined the effects of heptaphylline and 7-methoxyheptaphylline on apoptosis of human colon adenocarcinoma cells (HT-29 cell line). Quantification of cell viability was performed using cell proliferation assay (MTT assay) and of protein expression through immunoblotting. The results showed that only heptaphylline, but not 7-methoxyheptaphylline, significantly significantly activated cleaved of caspase-3 and poly (ADP-ribose) polymerase (PARP-1) which resulted in HT-29 cell death. We found that heptaphylline activated BH3 interacting-domain death agonist (Bid) and Bak, proapoptotic proteins. In contrast, it suppressed X-linked inhibitor-of-apoptosis protein (XIAP), Bcl-xL and survivin, inhibitors of apoptosis. In addition, heptaphylline inhibited activation of NF-κB/p65 (rel), a regulator of apoptotic regulating proteins by suppressing the activation of Akt and IKKα, upstream regulators of p65. The findings suggested that heptaphylline induces apoptosis in human colon adenocarcinoma cells .

Mechanistic Study of the Phytocompound, 2-β-D-Glucopyranosyloxy-1-hydroxytrideca-5,7,9,11-tetrayne in Human T-Cell Acute Lymphocytic Leukemia Cells by Using Combined Differential Proteomics and Bioinformatics Approaches

Bidens pilosa, a medicinal herb worldwide, is rich in bioactive polyynes. In this study, by using high resolution 2-dimensional gel electrophoresis coupled with mass spectrometry analysis, as many as 2000 protein spots could be detected and those whose expression was specifically up- or downregulated in Jurkat T cells responsive to the treatment with 2-β-D-glucopyranosyloxy-1-hydroxytrideca-5,7,9,11-tetrayne (GHTT) can be identified. GHTT treatment can upregulate thirteen proteins involved in signal transduction, detoxification, metabolism, energy pathways, and channel transport in Jurkat cells. Nine proteins, that is, thioredoxin-like proteins, BH3 interacting domain death agonist (BID protein involving apoptosis), methylcrotonoyl-CoA carboxylase beta chain, and NADH-ubiquinone oxidoreductase, were downregulated in GHTT-treated Jurkat cells. Further, bioinformatics tool, Ingenuity software, was used to predict signaling pathways based on the data obtained from the differential proteomics approach. Two matched pathways, relevant to mitochondrial dysfunction and apoptosis, in Jurkat cells were inferred from the proteomics data. Biochemical analysis further verified both pathways involving GHTT in Jurkat cells. These findings do not merely prove the feasibility of combining proteomics and bioinformatics methods to identify cellular proteins as key players in response to the phytocompound in Jurkat cells but also establish the pathways of the proteins as the potential therapeutic targets of leukemia.

Elevated Pressure Enhanced Trail-Induced Apoptosis in Hepatocellular Carcinoma Cells Via Erk1/2-Inactivation.

The high frequency of intrinsic resistance to TNF-related apoptosisinducing ligand (TRAIL) in tumor cell lines has necessitated the development of strategies to sensitize tumors to TRAIL-induced apoptosis. We previously showed that elevated pressure applied as a mechanical stressor enhanced TRAIL-mediated apoptosis in human lung carcinoma cells in vitro and in vivo. This study focused on the effect of elevated pressure on the sensitization of TRAIL-resistant cells and the underlying mechanism. We observed elevated pressure-induced sensitization to TRAIL-mediated apoptosis in Hep3B cells, accompanied by the activation of several caspases and the mitochondrial signaling pathway. Interestingly, the enhanced apoptosis induced by elevated pressure was correlated with suppression of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) phosphorylation and CREB without any change to other MAPKs. Phosphorylation of Bcl-2-associated death promoter (BAD) also decreased, leading to inhibition of the mitochondrial pathway. To confirm whether the activation of pERK1/2 plays a key role in the TRAIL-sensitizing effect of elevated pressure, Hep3B cells were pre-treated with the ERK1/2-specific inhibitor PD98059 instead of elevated pressure. Co-treatment with PD98059 and TRAIL augmented TRAIL-induced apoptosis and decreased BAD phosphorylation. The inhibition of ERK1/2 activation by elevated pressure and PD98059 also reduced BH3 interacting-domain death agonist (BID), thereby amplifying apoptotic stress at the mitochondrial level. Our results suggest that elevated pressure enhances TRAIL-induced apoptosis of Hep3B cells via specific suppression of ERK1/2 activation among MAPKs.

Association of BID SNPs (rs8190315 and rs2072392) and clinical features of benign prostate hyperplasia in Korean population.

Exercise has beneficial effect on cancer apoptosis and benign prostatic hyperplasia (BPH). The BH3 interacting domain death agonist (BID) gene expression is associated with apoptosis or cell proliferation. In this study, we investigated the association between BID single nucleotide polymorphisms (SNPs) and the development, prostate volume, and international prostate symptom score (IPSS) of BPH. In 222 BPH males and 214 controls, two SNPs in BID [rs8190315 (Ser56Gly), and rs2072392 (Asp106Asp)] were genotyped and analyzed using multiple logistic regression models. In the result, the genotype and allele frequencies of rs8190315 and rs2072392 were not associated with BPH development or IPSS, however, the allele frequencies [odd ratio (OR)= 1.90, 95% confidence interval (CI)= 1.07–3.41, P= 0.03] and genotype frequencies (in dominant model, OR= 1.94, 95% CI= 1.01–3.74, P= 0.42) of rs8190315, and the genotype frequencies of rs2072392 (in dominant model, OR= 1.94, 95% CI= 1.01–3.74, P= 0.42) were associated with increased prostate volume. We propose that rs8190315 and rs2072392 of BID may contribute to the disease severity of BPH.

Silencing of JMJD2B induces cell apoptosis via mitochondria-mediated and death receptor-mediated pathway activation in colorectal cancer.

To investigate the molecular mechanism of colorectal cancer (CRC) cell apoptosis induced by the Jumonji domain containing 2B (JMJD2B) silencing. Both HCT116 and LoVo CRC cell lines were used for analyses. Cell apoptosis after JMJD2B silencing was determined by flow cytometry. JC-1 fluorescence probe was applied to measure the mitochondrial outer membrane permeabilization by flow cytometry and fluorescence microscopy. Immunofluorescence was used to detect cytochrome C translocation from mitochondria to cytosol after JMJD2B silencing. The efficacy of JMJD2B silencing on the protein levels of Bcl-2 family, caspase proteins, CCAAT/enhancer binding protein homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) were detected by Western blot. JMJD2B silencing induced CRC cell apoptosis via a decrease of the anti-apoptotic gene Bcl-2 family expression, leading to the translocation of Bak and Bax proteins and the promotion of mitochondrial membrane disruption, resulting in the release of cytochrome C from mitochondria and subsequent caspase-9 and caspase-3 cleavage. It also increased the amount of cleaved caspase-8 involved in the death receptor-related apoptotic pathway. Bcl-2 homology 3 interacting-domain death agonist (Bid), a specific caspase-8 substrate involved in the Fas signaling pathway, subsequently induced cleavage via caspase-8 activation. However, levels of CHOP and GRP78 remained unchanged after JMJD2B silencing. JMJD2B silencing induced CRC cell apoptosis via both mitochondria-related and death receptor-related pathways. The cleavage of Bid activated by caspase-8 might serve as a crosstalk mediator between these two pathways in CRC.

B Cell Lymphoma-2 (BCL-2) Homology Domain 3 (BH3) Mimetics Demonstrate Differential Activities Dependent upon the Functional Repertoire of Pro- and Anti-apoptotic BCL-2 Family Proteins

The B cell lymphoma-2 (BCL-2) family is the key mediator of cellular sensitivity to apoptosis during pharmacological interventions for numerous human pathologies, including cancer. There is tremendous interest to understand how the proapoptotic BCL-2 effector members (e.g. BCL-2-associated X protein, BAX) cooperate with the BCL-2 homology domain only (BH3-only) subclass (e.g. BCL-2 interacting mediator of death, BIM; BCL-2 interacting-domain death agonist, BID) to induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis and whether these mechanisms may be pharmacologically exploited to enhance the killing of cancer cells. Indeed, small molecule inhibitors of the anti-apoptotic BCL-2 family members have been designed rationally. However, the success of these "BH3 mimetics" in the clinic has been limited, likely due to an incomplete understanding of how these drugs function in the presence of multiple BCL-2 family members. To increase our mechanistic understanding of how BH3 mimetics cooperate with multiple BCL-2 family members in vitro, we directly compared the activity of several BH3-mimetic compounds (i.e. ABT-263, ABT-737, GX15-070, HA14.1, TW-37) in biochemically defined large unilamellar vesicle model systems that faithfully recapitulate BAX-dependent mitochondrial outer membrane permeabilization. Our investigations revealed that the presence of BAX, BID, and BIM differentially regulated the ability of BH3 mimetics to derepress proapoptotic molecules from anti-apoptotic proteins. Using mitochondria loaded with fluorescent BH3 peptides and cells treated with inducers of cell death, these differences were supported. Together, these data suggest that although the presence of anti-apoptotic BCL-2 proteins primarily dictates cellular sensitivity to BH3 mimetics, additional specificity is conferred by proapoptotic BCL-2 proteins.

The serine protease inhibitor TLCK attenuates intrinsic death pathways in neurons upstream of mitochondrial demise.

It is well-established that activation of proteases, such as caspases, calpains and cathepsins are essential components in signaling pathways of programmed cell death (PCD). Although these proteases have also been linked to mechanisms of neuronal cell death, they are dispensable in paradigms of intrinsic death pathways, e.g. induced by oxidative stress. However, emerging evidence implicated a particular role for serine proteases in mechanisms of PCD in neurons. Here, we investigated the role of trypsin-like serine proteases in a model of glutamate toxicity in HT-22 cells. In these cells glutamate induces oxytosis, a form of caspase-independent cell death that involves activation of the pro-apoptotic protein BH3 interacting-domain death agonist (Bid), leading to mitochondrial demise and ensuing cell death. In this model system, the trypsin-like serine protease inhibitor Nα-tosyl-l-lysine chloromethyl ketone hydrochloride (TLCK) inhibited mitochondrial damage and cell death. Mitochondrial morphology alterations, the impairment of the mitochondrial membrane potential and ATP depletion were prevented and, moreover, lipid peroxidation induced by glutamate was completely abolished. Strikingly, truncated Bid-induced cell death was not affected by TLCK, suggesting a detrimental activity of serine proteases upstream of Bid activation and mitochondrial demise. In summary, this study demonstrates the protective effect of serine protease inhibition by TLCK against oxytosis-induced mitochondrial damage and cell death. These findings indicate that TLCK-sensitive serine proteases play a crucial role in cell death mechanisms upstream of mitochondrial demise and thus, may serve as therapeutic targets in diseases, where oxidative stress and intrinsic pathways of PCD mediate neuronal cell death.

JNK1/2 regulate Bid by direct phosphorylation at Thr59 in response to ALDH1L1.

BH3 interacting-domain death agonist (Bid) is a BH3-only pro-apoptotic member of the Bcl-2 family of proteins. Its function in apoptosis is associated with the proteolytic cleavage to the truncated form tBid, mainly by caspase-8. tBid translocates to mitochondria and assists Bax and Bak in induction of apoptosis. c-Jun N-terminal kinase (JNK)-dependent alternative processing of Bid to jBid was also reported. We have previously shown that the folate stress enzyme 10-formyltetrahydrofolate dehydrogenase (ALDH1L1) activates JNK1 and JNK2 in cancer cells as a pro-apoptotic response. Here we report that in PC-3 prostate cancer cells, JNK1/2 phosphorylate Bid at Thr59 within the caspase cleavage site in response to ALDH1L1. In vitro, all three JNK isoforms, JNK 1–3, phosphorylated Thr59 of Bid with JNK1 being the least active. Thr59 phosphorylation protected Bid from cleavage by caspase-8, resulting in strong accumulation of the full-length protein and its translocation to mitochondria. Interestingly, although we did not observe jBid in response to ALDH1L1 in PC-3 cells, transient expression of Bid mutants lacking the caspase-8 cleavage site resulted in strong accumulation of jBid. Of note, a T59D mutant mimicking constitutive phosphorylation revealed more profound cleavage of Bid to jBid. JNK-driven Bid accumulation had a pro-apoptotic effect in our study: small interfering RNA silencing of either JNK1/2 or Bid prevented Bid phosphorylation and accumulation, and rescued ALDH1L1-expressing cells. As full-length Bid is a weaker apoptogen than tBid, we propose that the phosphorylation of Bid by JNKs, followed by the accumulation of the full-length protein, delays attainment of apoptosis, and allows the cell to evaluate the stress and make a decision regarding the response strategy. This mechanism perhaps can be modified by the alternative cleavage of phospho-T59 Bid to jBid at some conditions.

Metformin inhibits the proliferation of A549/CDDP cells by activating p38 mitogen-activated protein kinase.

Metformin (Met) has been widely used in hypoglycemic therapy, and it is also able to reduce the incidence of tumors and tumor-related mortality. The present study investigated whether Met could inhibit the proliferation of lung cancer cells and enhance the sensitivity of a cisplatin-resistant lung cancer A549/CDDP cell line to cisplatin. It was found that Met treatment inhibited the proliferation of different lung cancer cells. Met inhibited the cell cycle of the A549/CDDP cells and induced apoptosis. Upon Met treatment, the A549/CDDP cells were arrested at the G1 phase. The apoptosis of the A549/CDDP cells was confirmed by the appearance of apoptotic bodies in cells stained with Hoechst 33258, and by the cleavage of BH3 interacting-domain death agonist and poly (ADP-ribose) polymerase. Furthermore, results showed that the phosphorylation level of p38 mitogen-activated protein kinase (MAPK) was increased after Met treatment. The p38 MAPK inhibitor, SB203580, significantly blocked Met-induced apoptosis in the A549/CDDP cells. It was further demonstrated that Met could enhance the sensitivity of the A549/CDDP cells to cisplatin. In summary, the present study identified Met as a drug sensitizer that could improve the treatment effect of cisplatin in cisplatin-resistant lung cancers.

Association of BH3 interacting domain death agonist (BID) gene polymorphisms with proteinuria of immunoglobulin A nephropathy

Background. Apoptosis plays an important role in the mechanism regulating the development of glomerulonephritis. We investigated whether polymorphisms of apoptotic genes such as B-cell CLL/lymphoma 2 (BCL2), BH3-interacting domain death agonist (BID), and caspase 8 (CASP8) were associated with immunoglobulin A nephropathy (IgAN) and with the clinical phenotypes of IgAN patients. Methods. We genotyped promoter and coding region single nucleotide polymorphisms (SNPs) (rs2279115 and rs1801018 for BCL2; rs8190315 and rs2072392 for BID; and rs6747918 and rs1045487 for CASP8) using direct sequencing in 195 IgAN patients and 289 control subjects. Results. No SNPs were associated with IgAN. However, in analysis of clinical phenotypes, we found that rs8190315 and rs2072392 of BID were associated with proteinuria levels of IgAN patients in additive (AG vs. GG vs. AA, p = 0.0008 for rs8190315; TC vs. CC vs. TT, p = 0.0012 for rs2072392) and dominant models (AG/GG vs. AA, p = 0.0014 for rs8190315; TC/CC vs. TT, p = 0.0031 for rs2072392). In particular, the frequencies of genotypes containing minor alleles of rs8190315 (G allele) and rs2072392 (C allele) were increased in IgAN patients with higher protienuria levels (> 40 mg/m2/h). Conclusion. These results suggest that BID may play a role in severe IgAN.

Association between BH3 interacting domain death agonist (BID) gene polymorphism and ossification of the posterior longitudinal ligament in Korean population

The purpose of this study was to investigate single nucleotide polymorphisms (SNPs) of the BH3 interacting domain death agonist (BID) gene as a risk factor in Korean patients with ossification of the posterior longitudinal ligament (OPLL). To investigate the genetic association, two coding SNPs (rs8190315, Ser10Gly; rs2072392, Asp60Asp) of BID were genotyped in 157 OPLL patients and 209 control subjects. SNPStats, SNPAnalyzer Pro, Helixtree, and Haploview 4.2 programs were used for association analysis. Multiple logistic regression models (codominant, dominant, and recessive) were calculated for the odds ratios (ORs), 95% confidence intervals (CIs), and corresponding P values. For multiple testing, Bonferroni correction was performed. After Bonferroni correction, genotype analysis of both rs8190315 and rs2072392 showed association between the OPLL group and the control group in the codominant model (P=0.042, OR 1.86, 95% CI 1.10-3.15). A complete linkage disequilibrium block was estimated between the two SNPs. Both of the G allele of rs8190315 and C allele of rs2072392 were strongly associated with an increased risk in the development of OPLL (P=0.0052, OR 2.66, 95% CI 1.51-4.68). These results suggest that BID is associated with OPLL, and both the G allele of a missense SNP (rs8190315, Ser10Gly) and C allele of a synonymous SNP (rs2072392, Asp60Asp) are risk factors for the development of OPLL in Korean population.

Three-dimensional structure of Bax-mediated pores in membrane bilayers

B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) is a member of the Bcl-2 protein family having a pivotal role in triggering cell commitment to apoptosis. Bax is latent and monomeric in the cytosol but transforms into its lethal, mitochondria-embedded oligomeric form in response to cell stress, leading to the release of apoptogenic factors such as cytochrome C. Here, we dissected the structural correlates of Bax membrane insertion while oligomerization is halted. This strategy was enabled through the use of nanometer-scale phospholipid bilayer islands (nanodiscs) the size of which restricts the reconstituted system to single Bax-molecule activity. Using this minimal reconstituted system, we captured structural correlates that precede Bax homo-oligomerization elucidating previously inaccessible steps of the core molecular mechanism by which Bcl-2 family proteins regulate membrane permeabilization. We observe that, in the presence of BH3 interacting domain death agonist (Bid) BH3 peptide, Bax monomers induce the formation of ∼3.5-nm diameter pores and significantly distort the phospholipid bilayer. These pores are compatible with promoting release of ions as well as proteinaceous components, suggesting that membrane-integrated Bax monomers in the presence of Bid BH3 peptides are key functional units for the activation of the cell demolition machinery.

Hypericin-photodynamic therapy leads to interleukin-6 secretion by HepG2 cells and their apoptosis via recruitment of BH3 interacting-domain death agonist and caspases

Photodynamic therapy (PDT) has emerged as a capable therapeutic modality for the treatment of cancer. PDT is a targeted cancer therapy that reportedly leads to tumor cell apoptosis and/or necrosis by facilitating the secretion of certain pro-inflammatory cytokines and expression of multiple apoptotic mediators in the tumor microenvironment. In addition, PDT also triggers oxidative stress that directs tumor cell killing and activation of inflammatory responses. However, the cellular and molecular mechanisms underlying the role of PDT in facilitating tumor cell apoptosis remain ambiguous. Here, we investigated the ability of PDT in association with hypericin (HY) to induce tumor cell apoptosis by facilitating the induction of reactive oxygen species (ROS) and secretion of Th1/Th2/Th17 cytokines in human hepatocellular liver carcinoma cell line (HepG2) cells. To discover if any apoptotic mediators were implicated in the enhancement of cell death of HY-PDT-treated tumor cells, selected gene profiling in response to HY-PDT treatment was implemented. Experimental results showed that interleukin (IL)-6 was significantly increased in all HY-PDT-treated cells, especially in 1 μg/ml HY-PDT, resulting in cell death. In addition, quantitative real-time PCR analysis revealed that the expression of apoptotic genes, such as BH3-interacting-domain death agonist (BID), cytochrome complex (CYT-C) and caspases (CASP3, 6, 7, 8 and 9) was remarkably higher in HY-PDT-treated HepG2 cells than the untreated HepG2 cells, entailing that tumor destruction of immune-mediated cell death occurs only in PDT-treated tumor cells. Hence, we showed that HY-PDT treatment induces apoptosis in HepG2 cells by facilitating cytotoxic ROS, and potentially recruits IL-6 and apoptosis mediators, providing additional hints for the existence of alternative mechanisms of anti-tumor immunity in hepatocellular carcinoma, which contribute to long-term suppression of tumor growth following PDT.

Apoptosis in TMJ Disc Diseases

The apoptosis is the programmed cell death, a distinct process compared to cellular necrosis, which plays an important role in both human embryonic development and adult tissue homeostasis. Apoptosis represents a physiological form of cell death. The role of apoptosis is to remove harmful, damaged or unwanted cells without inducing inflammatory response by the release of cell contents [1]. Apoptosis is activated through two principal signaling pathways: intrinsic and extrinsic, both of which are potential anticancer therapeutic targets. In contrast to necrosis, which is a form of cell death resulting from an acute cellular stress or trauma, apoptosis is a death orderly and regular, does not induce inflammation, requires energy (ATP) and generally lead to an advantage during the body life. Besides its importance as a biological phenomenon, it has acquired a huge medical value, since imperfect process of apoptosis covers many diseases. Excessive activity can cause trouble by apoptotic cell loss (see, for example neurodegenerative diseases such as Parkinson’s disease), while a weak apoptosis may involve uncontrolled cell growth in malignancy mechanism [2]. Apoptosis is the most common mechanism by which the body eliminates damaged or unneeded cells, without local inflammation from leakage of cell contents. Cells that are undergoing apoptosis exhibit a characteristic pattern of morphologic changes, including cell shrinkage, condensation, fragmentation of the nucleus and bubbling of the plasma membrane, known as “blebbing,” and chromatin condensation and nucleosomal fragmentation [1,3]. Apoptosis functions through two main, alternative pathways: death receptor-mediated (or extrinsic) and mitochondria-dependent (or intrinsic). The former pathway is initiated by ligation of specific death receptors by their ligands. The main death receptors—Fas and tumour necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) receptors R1 and R2—induce cell death following ligation with Fas ligand (FasL) or TRAIL, respectively [4-7]. Ligation of TRAIL R1 by TNFα also induces apoptosis after inhibition of nuclear factor kappa-B (NF-kB). Fas ligation by FasL is followed by recruitment of FADD (Fas-associated via death domain) and subsequently of caspase 8. This process gives rise to caspase 8 activation, which can be inhibited by the anti-apoptotic molecule FLICE inhibitory protein (Flip). Caspase 8 induces apoptosis by directly activating caspase 3 or by cleaving bid (BH3 interacting domain death agonist), resulting in mitochondrial dysfunction and subsequent release of cytochrome c and activation of caspases 9 and 3. Caspase 3 promotes the typical apoptosis features, including DNA fragmentation and cell death in several tissues [8,9]. It is unclear whether repetitive loading and apoptosis are quantitatively related in any type of cartilage. Several studies have demonstrated the crucial function of apoptotic mechanisms in intervertebral disc degeneration, and the involvement of apoptosis in various conditions associated with intervertebral disc degeneration has been thoroughly explored [10]. However, apoptosis has been documented to have a central role not only in intervertebral disc degeneration [10]. Although a number of features of temporomandibular joint (TMJ) disc degeneration have been thoroughly studied, data on the involvement of apoptotic mechanisms and their mediators are few and quite recent; indeed most of the research conducted on disc apoptosis has focused on the intervertebral disc. TMJ disc degeneration is believed to be a consequence of mechanical and biological events affecting the equilibrium between matrix synthesis and degradation. According to this hypothesis loss of cellularity, collagen fibre fragmentation and TMJ disc tears and clefts would all depend on excessive apoptosis, a situation where collagen fibre degradation is not offset by synthesis of new fibres because of apoptosis-induced cell loss. The findings correlating TMJ disc internal derangement and apoptosis largely come from animal models [11]. However, our group has extensively analyzed programmed cell death in human TMJ discs with and without internal derangement [12,13]. We have advanced Corresponding author.

AIF-mediated caspase-independent necroptosis requires ATM and DNA-PK-induced histone H2AX Ser139 phosphorylation

The alkylating DNA-damage agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces a form of caspase-independent necroptosis implicating the mitochondrial flavoprotein apoptosis-inducing factor (AIF). Following the activation of PARP-1 (poly(ADP-ribose) polymerase-1), calpains, BID (BH3 interacting domain death agonist), and BAX (Bcl-2-associated X protein), the apoptogenic form of AIF (tAIF) is translocated to the nucleus where, associated with Ser139-phosphorylated histone H2AX (γH2AX), it creates a DNA-degrading complex that provokes chromatinolysis and cell death by necroptosis. The generation of γH2AX is crucial for this form of cell death, as mutation of H2AX Ser139 to Ala or genetic ablation of H2AX abolish both chromatinolysis and necroptosis. On the contrary, reintroduction of H2AX-wt or the phosphomimetic H2AX mutant (H2AX-S139E) into H2AX−/− cells resensitizes to MNNG-triggered necroptosis. Employing a pharmacological approach and gene knockout cells, we also demonstrate in this paper that the phosphatidylinositol-3-OH kinase-related kinases (PIKKs) ATM (ataxia telangiectasia mutated) and DNA-dependent protein kinase (DNA-PK) mediate γH2AX generation and, consequently, MNNG-induced necroptosis. By contrast, H2AX phosphorylation is not regulated by ATR or other H2AX-related kinases, such as JNK. Interestingly, ATM and DNA-PK phosphorylate H2AX at Ser139 in a synergistic manner with different kinetics of activation. Early after MNNG treatment, ATM generates γH2AX. Further, DNA-PK contributes to H2AX Ser139 phosphorylation. In revealing the pivotal role of PIKKs in MNNG-induced cell death, our data uncover a milestone in the mechanisms regulating AIF-mediated caspase-independent necroptosis.