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The present research seeks to investigate the process of mixing targeted gene delivery and transcriptional targeting. We have conjugated Polyethylenimine polymers (PEI) and molecules of poly (ethylene glycol). The next step was covalent attachment of anti-HER2 variables domains of camelid heavy chains antibodies (VHHs) or nanobodies (Nbs) to the distal terminals of NHS-PEG3500 in PEI-PEG nanoparticles. The whole procedure yielded PEI-PEG-Nb immunoconjugates. Having determined the properties of polyplexes, steps were taken to investigate the most efficient ratio of PEI polymers to pDNA molecules (N/P) so that the greatest rate of transfection may be obtained. This immune targeted nano biopolymer could condense the gene constructs that coded a transcriptionally targeted truncated -Bid (tBid) killer gene which was controlled by the breast cancer-specific MUC1 promoter. The favourable physicochemical properties matching both the size and zeta potential were observed in engineered polyplexes. Elevated transfection efficiency in HER2 positive cell lines using Nb-modified polyplexes were shown by the results of flow cytometry as compared against non-modified particles. 1.6 and 4.8 fold higher transfection efficiencies were observed in in vitro gene expression researches which used PEI-PEG-Nb/pGL4.50 compared to the situation when native PEI polymers were utilized in both BT-474 and SK-BR-3, respectively. A 2.22 and 3.62 fold rise in the level of tBid gene expression in BT-474 and SK-BR-3 cell lines relative to unmodified PEI treated cells was the result of transfection with PEI-PEG-Nb/pMUC1-tBid, respectively. In those HER2-positive cells which were transfected by targeted polyplexes, higher levels of cell death were observed. This fact points not only to the effective targeted delivery, but it is also indicative of transcriptional targeting efficiency of tBid killer gene when its expression is controlled by MUC1 promoter.

The TNF-Related Apoptosis Inducing Ligand (TRAIL) cytokine triggers apoptosis specifically in cancer cells. Susceptibility of a given cell to TRAIL depends on the activity of regulatory proteins, one of the most important of which is BID. The aim of this study was to increase the cytotoxic potential of TRAIL against cancer cells. TRAIL was fused to the BH3 domain of BID. Hence, TRAIL acted not only as an anticancer agent, but also as a specific carrier for the BID fragment. Two fusion protein variants were obtained by genetic engineering, harboring two different linker sequences. The short linker allowed both parts of the fusion protein to fold into their native structures. The long linker influenced the structure of the fused proteins but nonetheless resulted in their highest cytotoxic activity. Optimal buffer formulation was determined for all the analyzed TRAIL variants. Fusing the BH3 domain of BID to TRAIL improved the cytotoxic potential of TRAIL. Further, these findings may be useful for the optimization of other anticancer drugs based on TRAIL, since the appropriate formulation would secure their native structures during prolonged storage., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objective: Previous studies found bone resorption and chondrocytes loss in mouse models of mid-palatal suture when given continuous compressive force, although chondrocytes response remained unknown. Herein, we design this study to determine how continuous compression force induces chondrocytes apoptosis., Methods: Thirty C57BL/6 male mice (aged 6 weeks) were randomly assigned into controls (not ligated to a spring), blank controls (ligated with no compression) and the compression group (ligated with 20-g compression). After 4 d, palatal tissues were sampled and stained by TB and safranin-O. Tunel staining measured the percentage of apoptotic chondrocytes, and immunohistochemistry was performed to label apoptosis-associated proteins (e.g., Bcl-2, Bcl-xl, Bax, Bak, Bid, Bad, caspase-3, caspase-8 and caspase-9). Intergroup comparison was made by the rank sum test, and P < 0.05 was defined as statistical significance., Results: After 7d of induction, TB and safranin-O staining revealed that the cartilage area in the compression group was significantly decreased, while the control group remained largely unaltered. Tunel staining showed that apoptotic cell numbers in the mid-palatal suture were significantly higher than the control group. Immunohistochemistry showed that mice in the compression group had significantly increased expression of caspase-3, caspase-9, Bad, Bak, Bax and Bid; However, caspase-8 remained unaltered. No expression of Bcl-2 and Bcl-xl was detected., Conclusions: Continuous compression force induces chondrocytes apoptosis in the mid-palatal suture. This process might be associated with the mitochondrial pathway., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Mesenchymal stem cells (MSCs) are promising vehicles for the delivery of anticancer agents in cancer therapy. However, the tumor targeting of loaded therapeutics is essential. Here, we explored a dual-targeting strategy to incorporate tumor-tropic MSC delivery with HER2-specific killing by the immunoapoptotin e23sFv-Fdt-tBid generated in our previous studies. The MSC engineering allowed simultaneous immunoapoptotin secretion and bioluminescence detection of the modified MSCs. Systemic administration of the immunoapoptotin-engineered MSCs was investigated in human HER2-reconstituted syngeneic mouse models of orthotopic and metastatic breast cancer, as well as in a xenograft nude mouse model of orthotopic gastric cancer. In vivo dual tumor targeting was confirmed by local accumulation of the bioluminescence-imaged MSCs and persistence of His-immunostained immunoapoptotins in tumor sites. The added tumor preference of MSC-secreted immunoapoptotins resulted in a significantly stronger antitumor effect compared with purified immunoapoptotins and Jurkat-delivered immunoapoptotins. This immunoapoptotin-armored MSC strategy provides a rationale for its use in extended malignancies by combining MSC mobility with redirected immunoapoptotins against a given tumor antigen., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Temporal post-conditioning helps provide neuroprotection against brain injury secondary to ischemia-reperfusion and is considered an effective intervention, but the exact mechanism of sevoflurane post-conditioning is unclear. The essential axis involves activator Bid, Bim, Puma (BH3s), Bax, and Bak; activates the mitochondrial death program; and might be involved in a cell death signal. Extracellular signal-related kinases 1/2 (Erk1/2) play a pivotal role in cell growth and proliferation. We hypothesized that sevoflurane post-conditioning might inhibit Bid, Bim, Puma, Bax, and Bak expression and is activated by phosphor-Erk1/2 to decrease neuronal death. To test this hypothesis, we exposed primary cortical neuron cultures to oxygen-glucose deprivation for 1h, along with resuscitation for 24h (OGD/R). MTT assays, propidium iodide uptake (PI), JC-1 fluorescence, and Western blot indicated the following: decreased cell viability (P<0.05); increased cell death (P<0.05); decreased mitochondrial membrane potential (P<0.05); and decreased Bid, Bim, Puma, Bax, and Bak expression with OGD/R exposure. Inhibition of Erk1/2 phosphorylation could attenuate sevoflurane post-conditioning that mediated an increase in neuronal viability and mitochondrial membrane potential, as well as a decrease in cell death and Bid, Bim, Puma, Bax, and Bak expression after OGD/R treatment. The results demonstrated that sevoflurane post-conditioning caused a marked decrease in cortical neuronal death secondary to OGD/R exposure through the downregulation of the mitochondrial apoptosis axis involving Bid, Bim, Puma, Bax, and Bak that was mediated by the phosphorylation/activation of Erk1/2., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Previous studies have suggested that the novel BH3 mimetic S1 could induce apoptosis in diverse tumor cell lines through endoplasmic reticulum (ER) stress or mitochondrial cell death pathways. The activation of c-Jun N-terminal kinase (JNK) through inositol requiring enzyme-1 (IRE1) is closely connected to ER stress-induced apoptosis. However, the role of JNK is complex, as there are different JNK subtypes and the function of each subtype is still not entirely clear. Here we found that the mRNA expression of JNK3 was continuously high in S1-treated human ovarian cancer SKOV3/DDP cells using a human unfolded protein response (UPR) pathway PCR array. Pharmacological inhibition of JNK3 increased cell sensitivity to apoptosis induced by S1. Furthermore, inhibition of JNK3 induced accumulation of both acidic compartment and p62, and upregulated ROS production. Our results suggest that JNK3 plays a pro-survival role during ER stress through preventing the block of autophagic flux and reducing oxidative stress in SKOV3/DDP cells. Inhibition of JNK3 may be a potential method to enhance the killing effect of the Bcl-2 inhibitor S1., (© 2014 Wiley Periodicals, Inc.)

Mesenchymal stem cells (MSC) secrete a great variety of cytokines that have beneficial paracrine actions. Hepatocyte growth factor (HGF) promotes proliferation in several cell types. The aim of the present study was to investigate the protective effect of HGF gene-transfected MSC (HGF-MSC) in acetaminophen (AAP)-treated hepatocytes. We transfected the HGF gene into MSCs and confirmed HGF expression by RT-PCR and western blot. The concentration of HGF in HGF-MSC conditioned media (HGFCM) was upregulated compared with that in control MSCCM samples. Cell viability was increased in HGFCM-treated hepatocytes. Expression of Mcl-1, an anti-apoptosis protein, was increased and expression of pro-apoptosis proteins (Bad, Bik and Bid) was decreased in HGFCM-treated hepatocytes. HGF-MSC had protective effects on AAP-induced hepatocyte damage by enhancing proliferation. These results suggest that HGF-expressing MSCs may provide regenerative potential for liver cell damage.

Unlabelled: In the present study we present data to show that certain tumor cells including malignant pleural mesothelioma (MPM) cells do not express argininosuccinate synthetase (ASS), and thus are unable to synthesize arginine from citrulline. Exposure of these ASS-negative cells to the arginine degrading enzyme, arginine deiminase (ADI-PEG20), for 72 h results in significant increases in cleaved caspase-3. Importantly, this apoptotic signal is further strengthened by the addition of TNF-related apoptosis-inducing ligand (TRAIL). Using flow cytometry, we showed that the combination treatment (ADI-PEG20 at 50 ng/ml and TRAIL at 10 ng/ml) for 24 h resulted in profound cell death with 67% of cells positive for caspase-3 activity, while ADI-PEG20 alone or TRAIL alone resulted in only 10-15% cell death. This positive amplification loop is mediated through the cleavage of proapototic protein "BID"., Conclusion: Our work represents a new strategy for treating patients with malignant pleural mesothelioma using targeted molecular therapeutics based on selected tumor markers, thus avoiding the use of potentially cytotoxic chemotherapy., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

The aim of the present study was to investigate and compare the effects of diferuloylmethane (curcumin) and diphenyldifluoroketone (EF-24) on cell growth and apoptosis induction in human osteogenic sarcoma cells. This was examined by MTT assay, nuclear DAPI staining, caspase-activation assay, flow cytometry analysis and immunoblotting in Saos2 human osteogenic sarcoma cells. Curcumin and EF-24 inhibited the growth of Saos2 cells in a dose-dependent manner. The apparent potency of EF-24 was more than 3-fold higher that of curcumin. Treatment with curcumin or EF-24 resulted in nuclear condensation and fragmentation in the cells. The caspase-3/-7 activities were detected in living cells treated with curcumin or EF-24. Flow cytometry showed that the rate of apoptosis was increased by curcumin and EF-24 compared to the control. Curcumin and EF-24 promoted the proteolytic cleavages of procaspase-3/-7/-8/-9 with increases in the amount of cleaved caspase-3/-7/-8/-9. The curcumin- or EF-24-induced apoptosis in the Saos2 cells was mediated by the expression of Fas and activation of caspase-8, caspase-3 and poly(ADP-ribose) polymerase. Immunoblotting revealed the Bid and Bcl-2 proteins to be downregulated, and truncated-Bid, Bax and p53 proteins to be upregulated by curcumin and EF-24. Curcumin and EF-24 increased the Bax/Bcl-2 ratio significantly. These results suggest that the curcumin and EF-24 inhibit cell proliferation and induce apoptotic cell death in Saos2 human osteogenic sarcoma cells via both the mitochondria-mediated intrinsic pathway and the death receptor-mediated extrinsic pathway, and may have potential properties for anti-osteosarcoma drug discovery.

Chronic infection with hepatitis B virus is a cause of end-stage liver disease and hepatocellular carcinoma (HCC). We previously screened fructose-bisphosphate aldolase B (ALDOB) as a candidate binding protein of hepatitis B surface antigen (HBsAg) using a yeast 2-hybrid assay. In this study we aimed to confirm ALDOB as a binding protein of the S region of the HbsAg (HBs) and to investigate the function and involved mechanism between its interactions during HCC development. Our results demonstrated that both of exogenous and endogenous ALDOB proteins bind to HBs and colocalize in the cytoplasm in vitro. The coexistence of HBs and ALDOB inhibit apoptosis of cisplatin-induced HepG2 cells. Furthermore, western blot analysis showed the coexistence of HBs and ALDOB enhance the phosphorylations of AKT and its downstream of GSK-3β (phosphorylation); decreased expression of the pro-apoptotic proteins Bax, Bid, Bim, and Puma; and increased expression of the prosurvival proteins Bcl-2, Bcl-xl, and Mcl-1 in HepG2 cells. These findings suggest that interaction between HBs and ALDOB might be applied as a potential therapeutic target during the treatment of HBV-related hepatitis or HCC.

This report is designed to explore the roles of caspase-8, -9 and -3 in artemisinin (ARTE)-induced apoptosis in non-small cell lung cancer cells (A549 cells). ARTE induced reactive oxygen species (ROS)-mediated apoptosis in dose- and time-dependent fashion. Although ARTE treatment did not induce Bid cleavage and significant loss of mitochondrial membrane potential, it induced release of Smac and AIF but not cytochrome c from mitochondria, and silencing of Bak but not Bax significantly prevented ARTE-induced cytotoxicity. Moreover, ARTE treatment induced ROS-dependent activation of caspase-9, -8 and -3. Of the utmost importance, silencing or inhibiting any one of caspase-8, -9 and -3 almost completely prevented ARTE-induced activation of all the three caspases and remarkably abrogated the cytotoxicity of ARTE, suggesting that ARTE triggered an amplification activation loop among caspase-9, -8 and -3. Collectively, our data demonstrate that ARTE induces a ROS-mediated amplification activation loop among caspase-9, -8 and -3 to dominantly mediate the apoptosis of A549 cells.

Experimental evidence indicates that prostate apoptosis response-4 (Par-4, also known as PAWR) is a key regulator of cancer cell survival and may be a target for cancer-selective targeted therapeutics. Par-4 was first identified in prostate cancer cells undergoing apoptosis. Both intracellular and extracellular Par-4 have been implicated in apoptosis. Relatively little is known about the role of Par-4 in breast cancer cell apoptosis. In this study, we sought to investigate the effects of Par-4 expression on cell proliferation, apoptosis and drug sensitivity in breast cancer cells. MCF-7 cells were stably transfected with expression vectors for Par-4, or transiently transfected with siRNA for Par-4 knockdown. Cell proliferation assays were performed using MTT and apoptosis was evaluated using acridine orange staining, fluorescence microscopy and flow cytometry. Par-4 overexpression reduced MCF-7 proliferation rates. Conversely, Par-4 knockdown led to increased MCF-7 proliferation. Par-4 downregulation also led to increased BCL-2 and reduced BID expression. Par-4 overexpression did not affect the cell cycle profile. However, MCF-7 cells with increased Par-4 expression showed reduced ERK phosphorylation, suggesting that the inhibition of cell proliferation promoted by Par-4 may be mediated by the MAPK/ERK1/2 pathway. MCF-7 cells with increased Par-4 expression showed a marginal increase in early apoptotic cells. Importantly, we found that Par-4 expression modulates apoptosis in response to docetaxel in MCF7 breast cancer cells. Par-4 exerts growth inhibitory effects on breast cancer cells and chemosensitizes them to docetaxel.

In chronic myeloid leukemia (CML), epigenetic modifications such as promoter hypermethylation and inactive histone modification are known mechanisms of drug resistance. In our study, we investigated the roles of promoter hypermethylation of BIM and BID genes and H3K27me3 histone modification on imatinib resistance. We detected higher expression levels of BIM and BID genes and lower expression levels of EZH2, EED2, SIRT1, and SUZ12 genes in imatinib-resistant K562/IMA-3 cells compared to imatinib-non-resistant K562 cells. While we determined the EZH2 and DNMT enzymes as bounded to the promoter of the BIM gene, we did not detect hypermethylation of this promoter. We also found the H3K27me3 histone modification promoter of BIM and BID genes in both cell lines. In conclusion, our results support the notion that DNA promoter methylation may be formed independently from EZH2-H3K27me3 and pro-apoptotic BIM and BID genes are not methyllated in the imatinib resistance of CML cells.

This study investigated tumor necrosis factor-α (TNF-α)-mediated death pathway contribution to hydroquinone (HQ) cytotoxicity in human leukemia U937 cells. HQ-induced apoptosis of human leukemia U937 cells was characterized by the increase in mitochondrial membrane depolarization, procaspase-8 degradation and tBid production. Downregulation of Fas-associated death domain protein (FADD) blocked HQ-induced procaspase-8 degradation and rescued the viability of HQ-treated cells, suggesting the involvement of a death receptor-mediated pathway in HQ-induced cell death. HQ induced increased TNF-α mRNA stability led to TNF-α protein expression upregulation, whereas HQ suppressed TNF-α-mediated NFκB pathway activation. HQ elicited protein phosphatase 2A catalytic subunit α (PP2Acα) upregulation via p38 mitogen-activated protein kinase (MAPK)-mediated CREB/c-Jun/ATF-2 phosphorylation, and PP2Acα upregulation was found to promote tristetraprolin (TTP) degradation. Suppression of p38 MAPK activation and protein phosphatase 2A (PP2A) activity abrogated TNF-α upregulation and procaspase degradation in HQ-treated cells. Overexpression of TTP suppressed HQ-induced TNF-α upregulation and restored the viability of HQ-treated cells. Moreover, TTP overexpression increased TNF-α mRNA decay in HQ-treated cells. Taken together, our data indicate that HQ elicits TNF-α upregulation via p38 MAPK/PP2A-mediated TTP downregulation, and suggest that the TNF-α-mediated death pathway is involved in HQ-induced U937 cell death. The same pathway was also proven to be involved in the HQ-induced death of human leukemia HL-60 and Jurkat cells.

Garlic (Allium sativum) compounds have recently received increasing attention due to their cancer chemopreventive properties, and their anticancer activities are extensively reported in many cancer cell lines. However, the anticancer activity and the signaling pathway associated with the induction of apoptosis by extracts of garlic cloves have not been elucidated. In this study, we examined the effects of hexane extracts of garlic cloves (HEGCs) on reactive oxygen species (ROS) production and the association of these effects with apoptotic cell death, using a Hep3B human hepatocarcinoma cell line in vitro. The results demonstrated that HEGCs mediate ROS production, and that this mediation is followed by a collapse of mitochondrial membrane potential (MMP, ΔΨm), the downregulation of anti-apoptotic Bcl-2 and Bcl-xL and the activation of caspase-9 and -3. HEGCs also promoted the activation of caspase-8 and the downregulation of Bid, a BH3-only pro-apoptotic member of the Bcl-2. However, the apoptotic phenomena displayed by HEGCs were significantly diminished by the presence of z-VAD-fmk (non-selective caspase inhibitor). Moreover, N-acetyl-L-cysteine (NAC), a widely used ROS scavenger, effectively blocked the HEGC-induced apoptotic effects via the inhibition of ROS production and MMP collapse. These observations clearly indicate that HEGC-induced ROS are key mediators of MMP collapse, which leads to the induction of apoptosis, followed by caspase activation.

Resveratrol (Res), from the skin of red grapes, induces apoptosis in some malignant cells, but there are no reports on the apoptotic effect of Res on human malignant pleural mesothelioma. We found that Res interacts with specificity protein 1 (Sp1). The IC50 for Res was 17 µM in MSTO-211H cells. Cell viability was decreased and apoptotic cell death was increased by Res (0-60 µM). Res increased the Sub-G1 population in MSTO-211H cells and significantly suppressed Sp1 protein levels, but not Sp1 mRNA levels. Res modulated the expression of Sp1 regulatory proteins including p21, p27, cyclin D1, Mcl-1 and survivin in mesothelioma cells. After treatment with Res, apoptosis signaling cascades were activated by the activation of Bid, Bim, caspase-3 and PARP, upregulation of Bax and downregulation of Bcl-xL. Res (20 mg/kg daily for 4 weeks) effectively suppressed tumor growth in vivo in BALB/c athymic (nu+/nu+) mice injected with MSTO-211H cells, an effect that was mediated by inhibition of Sp1 expression and induction of apoptotic cell death. Our results strongly suggest that Sp1 is a novel molecular target of Res in human malignant pleural mesothelioma.

Given that the studies on human small intestinal stromal tumors are very limited, our study was designed to determine the significance of apoptosis, the role of some apoptosis-related proteins, including Bax, Bad, Bid, Bcl-2, Bcl-Xl, and Bcl-W, in jejunal stromal tumors (JST). For this purpose, 52 samples were examined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and immunohistochemistry, including 40 cases from normal jejunum and 12 cases from the JST. The results showed that a significantly decreased apoptosis was observed in JST compared with normal jejunum (median of apoptotic index, 0.13% vs 15.17%, p < 0.001). In univariate analysis, statistically significant differences were shown in percentage of positive-expression of Bid (0.0% vs 77.5%, p < 0.001), Bcl-2 (91.7% vs 42.5%, p = 0.003), and Bcl-Xl (75.0% vs 35.0%, p = 0.014), but not in Bax, Bad, and Bcl-W. Moreover, Bcl-Xl was an independent parameter associated with JST by multivariate Logistic analysis. For the role of ratios of anti-apoptotic proteins to pro-apoptotic proteins, Bcl-Xl/Bid ratio was the most preferable index. In conclusion, decreased apoptosis and expression of Bid, increased level of Bcl-Xl may play some important roles in human JST, and Bcl-Xl/Bid ratio may be as a new potentially associated index, although they should be validated in more studies., (© 2011 The Authors. APMIS © 2011 APMIS.)

Many phytochemicals have been recognized to have potential therapeutic efficacy in cancer treatment. In this study, we investigated ethyl gallate (EG) for possible proapoptotic effects in the human promyelocytic leukemia cell line, HL-60. We examined cell viability, morphological changes, DNA content and fragmentation, and expression of apoptosis-related proteins for up to 48 h after EG treatment. The results showed that EG induced morphological changes and DNA fragmentation and reduced HL-60 cell viability in a dose-dependent and time-dependent manner. Western blotting analysis indicated that EG-mediated HL-60 apoptosis mainly occurred through the mitochondrial pathway, as shown by the release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (Endo G), as well as the upregulation of Bcl-2-associated X protein (Bax). EG also activated the death receptor-dependent pathway of apoptosis by enhancing the expression of caspases-8, -9, and -3 and the Bcl-2 interacting domain (Bid). Collectively, our results showed that EG induces apoptosis in HL-60 via mitochondrial-mediated pathways.

In mitochondrion-dependent type II apoptosis, BH3-interacting domain death agonist (BID) and BCL-2-associated X protein (BAX) promote death ligand and receptor-mediated cell death. In porcine ovaries, the levels of BID and BAX increase in follicular granulosa cells during atresia. In the present study, to confirm the pro-apoptotic activity of BID and BAX in granulosa cells, we examined the effect of RNA interference of BID or BAX on apoptosis using a human ovarian granulosa tumor cell line, KGN. By reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, expression of BID and BAX was detected in KGN cells. Then, we suppressed BID and BAX mRNA expression in KGN cells using small interfering RNA (siRNA). When BID or BAX was suppressed, a significant decrease in the apoptotic cell rate was noted. In granulosa-derived cells, BID and BAX showed pro-apoptotic activity. These results suggest that BID and BAX act as signal-transducing factors in mitochondrion-dependent type II apoptosis.

Previously, we had reported that overexpression of miR-23a~27a~24-2 cluster induces caspase-dependent and -independent apoptosis via JNK in human embryonic kidney (HEK293T) cells. Herein, we describe the molecular mechanism(s) responsible for miR-23a~27a~24-2 cluster induced apoptosis. Gene expression profiling was used to characterize the transcriptional response to miR-23a~27a~24-2 cluster overexpression in HEK293T cells. The microarray analysis gave 1,025 differentially expressed genes and analysis of the gene expression data with Ingenuity Pathway Analysis (IPA) software revealed p53 signaling, oxidative stress response and mitochondrial dysfunction among the top processes being affected. This data substantiates our previous study where we had reported increase of ROS and the release of proapoptotic factors such as cytochrome c (cyt c) and apoptosis-inducing factor (AIF) from the mitochondria to cytosol. Additionally, components of ER stress-mediated apoptosis pathway i.e., C/EBP homologous protein (CHOP/DDIT3/GADD153) and TRIB3, an Akt inhibitor were found to be significantly enriched. Also, the enhanced expression of ATF3 and ATF4 was observed at RNA as well as protein level in miR-23a~27a~24-2 cluster overexpressed HEK293T cells. Induction of BIM appeared to be specific, because ER stress caused only a minor change in the expression of the related BH3-only proteins BID or PUMA. The fact that miR-23a~27a~24-2 cluster triggered endoplasmic reticulum stress (ER stress) was further established by the increase in cytosolic calcium levels after overexpression of this cluster in HEK293T cells. These findings were also supported by PANTHER analysis wherein biological process categories of apoptosis and stress response were enriched. Taken together, this work underlines the role of ER stress in miR-23a~27a~24-2 cluster mediated apoptosis in HEK293T cells. Since the detailed knowledge of this cluster induced apoptosis has now been elucidated, the in vivo study of this cluster would help evaluate the prospect of its use as a therapeutic in diseases known to occur because of deregulation of apoptosis.

5-azacytidine (5AzC) is a cytidine analogue with two main effects on cellular conditions; DNA damage, resulting in apoptosis, and DNA hypomethylation, restoring normal growth control and differentiation. However, the molecular mechanism of 5AzC-induced apoptosis is not fully understood. The aim of the present study is to clarify this mechanism in mouse thymocytes in vivo. Ten-week-old, male C57BL/6J mice were injected with 5AzC (100 mg/kg) intraperitoneally, and thymuses were examined for apoptotic changes. In the 5AzC-treated thymus, increases of TUNEL-positive thymocytes and cleaved caspase-3 protein, both biochemical features of apoptosis, were detected. 5AzC-induced apoptosis was observed even in the thymuses of mice deficient in p53, a critical factor in the intrinsic apoptotic pathway, and mice with mutated Fas, a death receptor. Furthermore, levels of p53 and Fas proteins were unchanged in the thymus following 5AzC-treatment in wild-type mice. In the 5AzC-treated thymus, the level of cleaved caspase-8 protein, an initiator of the extrinsic apoptotic pathway, increased with the cleavage of its target protein, Bid. Moreover, the level of TRAIL protein, which induces apoptosis through the cleavage of caspase-8, robustly increased in the thymus treated with 5AzC. In conclusion, the 5AzC-induced apoptosis of thymocytes in vivo is implemented through the extrinsic pathway with the activation of TRAIL., (Copyright © 2009 Elsevier GmbH. All rights reserved.)

Francisella tularensis is a facultative intracellular, gram-negative bacterium that induces apoptosis in macrophages and B cells. Here we show apoptotic pathways that are activated in the Ramos human B cell line in the course of F. tularensis infection. Live bacteria F. tularensis FSC200 activate caspases 8, 9 and 3, as well as Bid; release cytochrome c and apoptosis-inducing factor from mitochondria; and induce depolarization of mitochondrial membrane potential in the Ramos cell line, thus leading these cells to apoptosis. Unlike live bacteria, killed F. tularensis FSC200 bacteria activated only caspase 3, and did not cause apoptosis of Ramos cells as measured by annexin V. Killed bacteria also caused accumulation of anti-apoptotic protein Bclx(L) in mitochondrial membranes. Thus, live F. tularensis activates both caspase pathways (receptor-mediated and intrinsic) as well as caspase-independent mitochondrial death., (Copyright 2010. Published by Elsevier India Pvt Ltd.)

Epithelial ovarian cancer (EOC) cells often show increased activity of the PI3K/Akt pathway. In addition, we have previously shown that EOC ascites induce Akt activation in the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitive EOC cell line, CaOV3, leading to TRAIL-mediated apoptosis inhibition. In this study, we investigated the role of Akt in intrinsic resistance to TRAIL, which is common in EOC cells. We report that Akt activation reduces the sensitivity of EOC cells to TRAIL. TRAIL-resistant SKOV3ip1 and COV2 cells were sensitized to TRAIL-induced apoptosis by PI3K or Akt inhibitors although inhibition of PI3K/Akt signaling pathway did not interfere with the recruitment and processing of caspase-8 to the death-inducing signaling complex. Conversely, overexpression of Akt1 in TRAIL-sensitive cells promoted resistance to TRAIL. Although the fact that TRAIL-induced caspase-8 activation was observed in both sensitive and resistant cell lines, Bid cleavage occurred only in sensitive cells or in SKOV3ip1 cells treated with LY294002. Bid expression was low in resistant cells and Akt activation downregulated its expression. Depletion of Bid by siRNA in OVCAR3 cells was associated with a decrease in TRAIL-mediated apoptosis. Overexpression of Bid only in SKOV3ip1 cells enhanced TRAIL-induced apoptosis. Simultaneous blockade of Akt pathway further increased TRAIL-induced apoptosis. Thus, Akt acts upstream of mitochondria and inhibits TRAIL-induced apoptosis by decreasing Bid protein levels and possibly inhibiting its cleavage.

Excessive fluoride ingestion has been identified as a risk factor for fluorosis and oxidative stress. The oxidative stress results from the loss of equilibrium between oxidative and antioxidative mechanisms that can produce kinase activation, mitochondrial disturbance and DNA fragmentation, resulting in apoptosis. Actually many people are exposed to no-adverted fluoride consumption in acute or chronic way. The aim of this study was to determine the effect of sodium fluoride on first molar germ in relation to its effect on antioxidative enzymes immunoexpression and apoptosis. Thirty first molar germs from 1-day-old Balb/c mice were cultured for 24 h with sodium fluoride (0 mM, 1 mM and 5 mM). Immunoexpression determination of CuZnSod, MnSod, catalase, Bax, Bid, caspase 8, caspase 9, caspase 3 and TUNEL assay were performed. Cellular disorganization in ameloblast and odontoblast-papilla zones was observed. CuZnSod and MnSod immunoexpression decrease in experimental groups. Caspase 8, caspase 3, Bax, Bid increase expression and more TUNEL positive cells in both experimental groups than control, suggest that apoptosis induced by fluoride is related to oxidative stress due to reduction of the enzymatic antioxidant., (© 2010 John Wiley & Sons A/S.)

Chronic lymphocytic leukemia (CLL) involves a profound humoral immune defect and tumor-specific humoral tolerance that directly contribute to disease morbidity and mortality. CD154 gene therapy can reverse this immune defect, but attempts to do this pharmacologically have been unsuccessful. The immune-modulatory agent lenalidomide shows clinical activity in CLL, but its mechanism is poorly understood. Here, we demonstrate that lenalidomide induces expression of functional CD154 antigen on CLL cells both in vitro and in vivo. This occurs via enhanced CD154 transcription mediated by a Nuclear Factor of Activated T cells c1 (NFATc1)/Nuclear Factor-kappaB (NF-kappaB) complex and also through phosphoinositide-3 (PI3)-kinase pathway-dependent stabilization of CD154 mRNA. Importantly, CD154-positive CLL cells up-regulate BID, DR5, and p73, become sensitized to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis, and promote costimulatory activation of normal B cells to produce antibodies. In CLL patients receiving lenalidomide, similar evidence of CD154 activation is observed including BID, DR5, and p73 induction and also development of anti-ROR1 tumor-directed antibodies. Our data demonstrate that lenalidomide promotes CD154 expression on CLL cells with subsequent activation phenotype, and may therefore reverse the humoral immune defect observed in this disease. This study is registered at http://clinicaltrials.gov as NCT00466895.

Developing cancer gene therapy constructs based on transcriptional targeting of genes to cancer cells is a new and promising modality for treatment of cancer. Introducing truncated Bid (tBid), a recently known member of the Bcl-2 family, eradicates cancer cells efficiently. For transcriptional targeting of tBid, two dual-specificity promoters, combining cancer specific core promoters and response modules, were designed. These two core promoter modules contained cancer specific promoters of MUC1 and Survivin genes accompanied by hypoxia-responsive elements and estrogen responsive elements (microenvironment condition of breast cancer cells) which were employed to achieve a higher and more specific level of tBid expression in breast cancer cells. Correlation of the level of tBid expression in normal and cancer cell lines with promoter activity was measured by RT-PCR after treatment with hypoxia and estrogen. The level of tBid expression under control of new hybrid promoters was compared with its expression under control of cytomegalovirus (CMV) promoter as a control. Our data revealed that the level of tBid expression in breast cancer cells were nearly 11 times more than normal cells because of the cancer specific promoters, although tBid expression under control of CMV promoter was almost the same in normal and cancer cell lines. Increased apoptosis was detected in the transfected breast cancer cell lines by the Caspase-3 activity assay. The application of these promoters may prove to have the advantage of tumor selective gene therapy in breast cancer cells and low-potential toxicity for normal tissues.

Ebolavirus (EBOV) is a member of the filovirus family and causes severe hemorrhagic fever, resulting in death in up to 90% of infected humans. EBOV infection induces massive bystander lymphocyte apoptosis; however, neither the cellular apoptotic pathway(s) nor the systemic implications of lymphocyte apoptosis in EBOV infection are known. In this study, we show data suggesting that EBOV-induced lymphocyte apoptosis in vivo occurs via both the death receptor (extrinsic) and mitochondrial (intrinsic) pathways, as both Fas-associated death domain dominant negative transgenic mice and mice overexpressing bcl-2 were resistant to EBOV-induced lymphocyte apoptosis. Surprisingly, inhibiting lymphocyte apoptosis during EBOV infection did not result in improved animal survival. Furthermore, we show for the first time that hepatocyte apoptosis likely occurs in EBOV infection, and that mice lacking the proapoptotic genes Bim and Bid had reduced hepatocyte apoptosis and liver enzyme levels postinfection. Collectively, these data suggest that EBOV induces multiple proapoptotic stimuli and that blocking lymphocyte apoptosis is not sufficient to improve survival in EBOV infection. These data suggest that hepatocyte apoptosis may play a role in the pathogenesis of EBOV infection, whereas lymphocyte apoptosis appears to be nonessential for EBOV disease progression.

The death of one cell can precipitate the death of nearby cells in a process referred to as the bystander effect. We investigated whether mitochondrial apoptosis generated a bystander effect and, if so, by which pathway. Microinjection with cytochrome c mimicked function of the mitochondrial apoptosis-induced channel MAC and caused apoptosis of both target and nearby osteoblasts. This effect was suppressed by inhibiting gap junction intercellular communication. A bystander effect was also observed after exogenous expression of tBid, which facilitates MAC formation and cytochrome c release. Interestingly, in connexin-43 deficient osteoblasts, microinjection of cytochrome c induced apoptosis only in the target cell. These findings indicate that a death signal was generated downstream of MAC function and was transmitted through gap junctions to amplify apoptosis in neighboring cells. This concept may have implications in development of new therapeutic approaches.

Heparin and low molecular weight heparins may reduce brain damage evoked by ischaemia/reperfusion (I/R) injury, although their use is hampered by the risk of haemorrhage. Chemical and enzymatic modifications of K5 polysaccharide have shown the possibility to produce heparin-like compounds with low anticoagulant activity and strong anti-inflammatory effects. Using a rat model of transient cerebral I/R, we investigated the effects of an epimerised N-,O-sulfated K5 polysaccharide derivative, K5-N,OSepi, on the infarct size, motor activity and injury caused by ischaemia (30 min) and reperfusion. Reperfusion was allowed for 60 min or 1-5 days. Rats reperfused for 5 days showed an infarct volume of 30.7 +/- 3.1% and K5-N,OSepi (0.1-1 mg/kg) caused dose-dependent reduction in infarct size (maximum at 1 mg/kg: 13.1 +/- 2.1% infarct volume). This effect was associated with a significant improvement in motor performance. In the rat hippocampus, one of the brain areas most sensitive to I/R injury, I/R induced a robust increase in myeloperoxidase (MPO) activity, a marker of neutrophil infiltration, that was halved by K5-N,OSepi administration (66.38 +/- 7.75 microU MPO/tissue g, 30.78 +/- 5.67 microU MPO/tissue g, respectively). K5-N,OSepi drastically reduced the expression of cyclooxygenase-2, inducible-nitric-oxide-synthase and intercellular-adhesion-molecule-1. I/R-induced activation of nuclear factor-kB was attenuated by drug treatment. Furthermore, K5-N,OSepi administration was associated with a significant modulation of apoptosis markers, such as Bid and Bcl-2. In conclusion, the results demonstrated that the sulfated semi-synthetic K5 derivative K5-N,OSepi protects the brain against I/R injury by disrupting multiple levels of the apoptotic and inflammatory cascade, including inhibition of NF-kappaB activation.

Large granular lymphocyte (LGL) leukemia is a clonal proliferative disease of T and natural killer (NK) cells. Interleukin (IL)-15 is important for the development and progression of LGL leukemia and is a survival factor for normal NK and T memory cells. IL-15 alters expression of Bcl-2 family members, Bcl-2, Bcl-XL, Bim, Noxa, and Mcl-1; however, effects on Bid have not been shown. Using an adoptive transfer model, we show that NK cells from Bid-deficient mice survive longer than cells from wild-type control mice when transferred into IL-15-null mice. In normal human NK cells, IL-15 significantly reduces Bid accumulation. Decreases in Bid are not due to alterations in RNA accumulation but result from increased proteasomal degradation. IL-15 up-regulates the E3 ligase HDM2 and we find that HDM2 directly interacts with Bid. HDM2 suppression by short hairpin RNA increases Bid accumulation lending further support for HDM2 involvement in Bid degradation. In primary leukemic LGLs, Bid levels are low but are reversed with bortezomib treatment with subsequent increases in LGL apoptosis. Overall, these data provide a novel molecular mechanism for IL-15 control of Bid that potentially links this cytokine to leukemogenesis through targeted proteasome degradation of Bid and offers the possibility that proteasome inhibitors may aid in the treatment of LGL leukemia.

The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is important in nasopharyngeal carcinoma (NPC) pathogenesis. Activated PI3K and its downstream target Akt are concernful signaling molecules and key survival factors involved in the control of cell proliferation, apoptosis and oncogenesis. The protein kinase Akt1, one of the Akt family members, is involved in several signaling pathways for tumor development and progression, suggesting that Akt1 might be an interesting target for a molecular tumor therapy. DNAzyme is a single-stranded DNA catalyst that can be engineered to bind to their complementary sequence in the target mRNA and cleave the mRNA. In this study, based on the analysis of sequences, thermodynamics and site distribution within the Akt1 gene, five DNAzymes were designed. We found that the DNAzyme, namely Dz2, strongly inhibited Akt1 mRNA and protein expression in the NPC cell line CNE1-LMP1. It was showed that the inhibition of cell proliferation, stimulation of apoptosis and inhibition of xenograft growth in nude mice can also exerted by Dz2. Besides, the apoptosis was shown to be associated with the suppression of the Bcl-2 and increased expression of Bax. Thus, DNAzyme targeting Akt1, Dz2, can inhibit multiple key tumorigenic processes in vitro and in vivo and may serve as a useful anti-cancer agent in NPC.

Human embryonic stem cells (hESCs) replicate in vitro by the process of self-renewal, whilst maintaining their pluripotency. Understanding the pathways involved in the regulation of this process will assist in developing fully-defined conditions for the robust proliferation of hESCs necessary for therapeutic applications. We previously demonstrated that sphingosine-1-phosphate (S1P) plays an important role in survival and proliferation of hESCs. and here the key signaling pathways and downstream targets of S1P were investigated in a representative cell line (Shef 4). A significant rise in ERK1/2 activation with S1P treatment was witnessed in hESCs maintained on murine embryonic fibroblasts (MEFs) exhibiting significantly higher levels of active ERK1/2 than those grown on Matrigel. RT-PCR and microarray analysis of micro-dissected, non-differentiated hESC revealed 1049 differentially expressed genes in S1P treated preparations compared with controls (n = 3). S1P regulated apoptosis through several BCL-2 family members, including BAX and BID, with increased expression of cell cycle progression genes associated with proliferation of hESC cultures. S1P treatment also increased expression of cell adhesion genes specifically cadherins and integrins. However, gene expression associated with pluripotency was decreased with S1P treatment indicating that an increased rate of hESC turnover (higher proliferation and lower apoptosis) may be balanced by an increased susceptibility to differentiate.

Dysregulation of professional APC has been postulated as a major mechanism underlying Ag-specific T cell hyporesponsiveness in patients with patent filarial infection. To address the nature of this dysregulation, dendritic cells (DC) and macrophages generated from elutriated monocytes were exposed to live microfilariae (mf), the parasite stage that circulates in blood and is responsible for most immune dysregulation in filarial infections. DC exposed to mf for 24-96 h showed a marked increase in cell death and caspase-positive cells compared with unexposed DC, whereas mf exposure did not induce apoptosis in macrophages. Interestingly, 48-h exposure of DC to mf induced mRNA expression of the proapoptotic gene TRAIL and both mRNA and protein expression of TNF-alpha. mAb to TRAIL-R2, TNF-R1, or TNF-alpha partially reversed mf-induced cell death in DC, as did knocking down the receptor for TRAIL-R2 using small interfering RNA. The mf also induced gene expression of BH3-interacting domain death agonist and protein expression of cytochrome c in DC; mf-induced cleavage of BH3-interacting domain death agonist could be shown to induce release of cytochrome c, leading to activation of caspase 9. Our data suggest that mf induce DC apoptosis in a TRAIL- and TNF-alpha-dependent fashion.

Purpose: Proapoptotic BH3-only proteins Bad and Bid initiate apoptosis by binding to regulatory sites on prosurvival Bcl-2 proteins to directly neutralize their function. We determined if expression of these proteins in colon cancers may account for differences in patient survival., Experimental Design: Tumor-node-metastasis stages II and III primary colon carcinomas from patients treated in 5-fluorouracil-based adjuvant therapy trials were studied. Immunohistochemical analysis of Bad and Bid proteins was done in tumors (n = 379) and adjacent normal mucosa. Expression was correlated with clinicopathologic variables, disease-free survival rates (DFS), and overall survival (OS) rates., Results: High expression of the Bad protein [hazard ratio (HR), 0.64; 95% confidence interval (95% CI), 0.43-0.96; P = 0.031] in the cytoplasm of tumor cells was significantly associated with more favorable OS in a univariate analysis. The combined Bad and Bid variable was prognostic for DFS (P = 0.027) and OS (P = 0.006). Stage and histologic grade, but not DNA mismatch repair status, were also prognostic for OS. Multivariate Cox analysis showed that high expression of Bad (HR, 0.64; 95% CI, 0.43-0.97; P = 0.027) and Bid (HR, 0.68; 95% CI, 0.49-0.97; P = 0.034) were independent predictors of OS after adjustment for stage, grade, age, treatment, and study. The combined variable of Bad + Bid was independently associated with DFS (P = 0.020) and OS (P = 0.004)., Conclusion: Proapoptotic Bad and Bid proteins are independent prognostic variables in colon cancer patients receiving adjuvant treatment. If validated, Bad and Bid expression may assist in risk stratification and selection of patients to receive adjuvant chemotherapy.

Aim: The possible enhancing effect of the combined use of sodium butyrate (SB) and hyperthermia to kill HCT 116 cells was evaluated., Materials and Methods: HCT 116 cells were subjected to SB (1 mM) treatment followed by hyperthermia (44 degrees C 60 min) and the effects on cell death, cell proliferation and the cell cycle were examined. Apoptosis-indicating protein expressions and intracellular superoxide formation were also analysed., Results: A marked reduction in the growth rate of the combined-treatment group was observed compared to those of the single-treatment groups. This involved the increased expression of p53 and p21, the alteration of the balance of anti- and proapoptotic Bcl-2 family proteins and enhanced superoxide formation. However, the death receptor pathway played no role., Conclusion: Hyperthermia synergistically promoted cell death induced by SB. Thus, the combined treatment led to mutual potentiation of the killing effects of each agent.

Peptide antibiotics possess the potent antimicrobial activities against invading microorganisms and contribute to the innate host defense. Antimicrobial human beta-defensins (hBDs) not only exhibit potent bactericidal activities against Gram-negative and Gram-positive bacteria but also function as immunomodulatory molecules by inducing cytokine and chemokine production and inflammatory and immune cell activation. Neutrophil is a critical effector cell in host defense against microbial infection, and its lifespan is regulated by various pathogen- and host-derived substances. Here, to further evaluate the role of hBDs in innate immunity, we investigated the action of hBD-1 to -4 on neutrophil apoptosis. Neutrophil apoptosis was assessed using human blood neutrophils based on the morphological changes. Of note, hBD-3 most potently suppressed neutrophil apoptosis among hBD-1 to -4, accompanied with the down-regulation of truncated Bid (a pro-apoptotic protein), up-regulation of Bcl-x(L) (an anti-apoptotic protein) and inhibition of mitochondrial membrane potential change and caspase 3 activity. Furthermore, we revealed that neutrophils expressed CC chemokine receptor (CCR) 6, and the action of hBD-3 was completely abrogated by a neutralizing anti-CCR6 mAb. Collectively, these observations suggest that hBDs, especially hBD-3, can not only kill bacteria but also modulate (suppress) neutrophil apoptosis via the action on CCR6. Suppression of neutrophil apoptosis results in the prolongation of their lifespan and may be advantageous for the host defense against bacterial invasion.

Quinolones (QNs)-induced arthropathy is an important toxic effect in immature animals leading to restriction of their therapeutic use in pediatrics. However, the exact mechanism still remains unclear. Recently, we have demonstrated that ofloxacin, a typical QN, induces apoptosis of alginate microencapsulated juvenile rabbit joint chondrocytes by disturbing the beta 1 integrin functions and inactivating the ERK/MAPK signaling pathway. In this study, we extend our initial observations to further elucidate the mechanism(s) of ofloxacin-induced apoptosis by utilizing specific caspase inhibitors. Pretreatment with both caspase-9-specific inhibitor zLEHD-fmk and caspase-8 inhibitor zIETD-fmk attenuated ofloxacin-induced apoptosis and activation of caspase-3 of chondrocyte in a concentration-dependent manner, as determined by fluorescent dye staining, enzyme activity assay and immunoblotting. Furthermore, the activation of caspase-9, -8 and -3 stimulated by ofloxacin was significantly inhibited in the presence of zIETD-fmk while pretreatment with zLEHD-fmk only blocked the activation of caspase-9 and -3. Ofloxacin also stimulated a concentration-dependent translocation of cytochrome c from mitochondria into the cytosol and a decrease of mitochondrial transmembrane potential, which was completely inhibited by zIETD-fmk. In addition, ofloxacin was found to increase the level of Bax, tBid, p53 in a concentration- and time-dependent manner. Taken together, The current results indicate that the caspase-8-dependent mitochondrial pathway is primarily involved in the ofloxacin-induced apoptosis of microencapsulated juvenile rabbit joint chondrocytes.

Introduction: In transgenic animal models of sepsis, members of the Bcl-2 family of proteins regulate lymphocyte apoptosis and survival of sepsis. This study investigates the gene regulation of pro-apoptotic and anti-apoptotic members of the Bcl-2 family of proteins in patients with early stage severe sepsis., Methods: In this prospective case-control study, patients were recruited from three intensive care units (ICUs) in a university hospital. Sixteen patients were enrolled when they fulfilled the criteria of severe sepsis. Ten critically ill but non-septic patients and 11 healthy volunteers served as controls. Blood samples were immediately obtained at inclusion. To confirm the presence of accelerated apoptosis in the patient groups, caspase-3 activation and phosphatidylserine externalisation in CD4+, CD8+ and CD19+ lymphocyte subsets were assessed using flow cytometry. Specific mRNAs of Bcl-2 family members were quantified from whole blood by real-time PCR. To test for statistical significance, Kruskal-Wallis testing with Dunn's multiple comparison test for post hoc analysis was performed., Results: In all lymphocyte populations caspase-3 (p < 0.05) was activated, which was reflected in an increased phosphatidylserine externalisation (p < 0.05). Accordingly, lymphocyte counts were decreased in early severe sepsis. In CD4+ T-cells (p < 0.05) and B-cells (p < 0.001) the Bcl-2 protein was decreased in severe sepsis. Gene expression of the BH3-only Bim was massively upregulated as compared with critically ill patients (p < 0.001) and 51.6-fold as compared with healthy controls (p < 0.05). Bid was increased 12.9-fold compared with critically ill patients (p < 0.001). In the group of mitochondrial apoptosis inducers, Bak was upregulated 5.6-fold, while the expression of Bax showed no significant variations. By contrast, the pro-survival members Bcl-2 and Bcl-xl were both downregulated in severe sepsis (p < 0.001 and p < 0.05, respectively)., Conclusions: In early severe sepsis a gene expression pattern with induction of the pro-apoptotic Bcl-2 family members Bim, Bid and Bak and a downregulation of the anti-apoptotic Bcl-2 and Bcl-xl proteins was observed in peripheral blood. This constellation may affect cellular susceptibility to apoptosis and complex immune dysfunction in sepsis.

Previous studies have suggested that Mcl-1, an antiapoptotic Bcl-2 homolog that does not exhibit appreciable affinity for the caspase 8-generated C-terminal Bid fragment (tBid), diminishes sensitivity to tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL). This study was performed to determine the mechanism by which Mcl-1 confers TRAIL resistance and to evaluate methods for overcoming this resistance. Affinity purification/immunoblotting assays using K562 human leukemia cells, which contain Mcl-1 and Bcl-x(L) as the predominant antiapoptotic Bcl-2 homologs, demonstrated that TRAIL treatment resulted in binding of tBid to Bcl-x(L) but not Mcl-1. In contrast, TRAIL caused increased binding between Mcl-1 and Bak that was diminished by treatment with the caspase 8 inhibitor N-(N(alpha)-acetylisoleucylglutamylthreonyl) aspartic acid (O-methyl ester)-fluoromethyl ketone (IETD(OMe)-fmk) or the c-Jun N-terminal kinase inhibitor SP600125. In addition, TRAIL caused increased binding of Bim and Puma to Mcl-1 that was inhibited by IETD(OMe)-fmk but not SP600125. Further experiments demonstrated that down-regulation of Mcl-1 by short hairpin RNA or the kinase inhibitor sorafenib increased TRAIL-induced Bak activation and death ligand-induced apoptosis in a wide variety of neoplastic cell lines as well as clinical acute myelogenous leukemia specimens. Collectively, these observations not only suggest a model in which Mcl-1 confers TRAIL resistance by serving as a buffer for Bak, Bim, and Puma, but also identify sorafenib as a potential modulator of TRAIL sensitivity.

Objective: To investigate the role of reactive oxygen species (ROS) and ROS-caused mitochondrial transmembrane potential loss in sodium selenite-induced apoptosis in NB4 cells., Methods: ROS production was measured by ROS-specific probe DCFH-DA. Sodium selenite mitochondrial transmembrane potential loss was evaluated by flow cytometry with Rh123 staining. Protein levels of cytochrome C, Bid, Bcl-xl, and Bax were measured by Western blot using protein-specific antibodies. NB4 cells were pre-incubated by MnTmPy or BSO before selenite treatment to further confirm the effects of ROS on NB4 cells., Results: 20 micromol/L sodium selenite induced ROS production and mitochondrial transmembrane potential loss in NB4 cells time-dependently. Cytochrome C accumulated in cytoplasm after selenite treatment. Sodium selenite also downregulated Bcl-xl and activated Bax and Bid at protein level. Pretreatment with antioxidant MnTmPy almost fully abrogated the proapoptotic effect of sodium selenite prevented the cleavage of Bid protein and in turn the mitochondrail transmembrane potential loss. On the contrary, pretreatment with BSO intensified the mitochondrail transmembrane potential loss induced by sodium selenite., Conclusions: Sodium selenite may induce apoptosis by inducing ROS production in NB4 cells, which leads to the downregulation of Bcl-xl, upregulation of Bax, and cleavage and activation of Bid. Bax and tBid then agregate on mitochondrial membrane, which in turn causes a decrease of mitochondrial transmembrane potential and release of cytochrome C into cytoplasm.

Microcystin-LR (MC-LR) produced by cyanobacteria in diverse water systems is a potent specific hepatotoxin and has been documented to induce hepatocyte apoptosis and liver injury; however, the mechanisms have not been fully elucidated. In the present study, we investigated whether MC-LR stimulated ROS generation in the liver of mice and the role of ROS in the pathogenesis of MC-LR-induced liver injury in vivo. MC-LR treatment (60 microg/kg of body weight) for 12h prompted large amount of ROS generation in mice liver, upregulated the expression of Bax and Bid, caused the mitochondrial membrane potential (MMP) loss and hepatocyte apoptosis as well as liver injury. While pretreatment with antioxidants, oral administration of vitamin C (250mg/kg of body weight, dissolved in double distill water) and vitamin E (200mg/kg of body weight, dissolved in corn oil) per day for 3 days continually, significantly reduced the generation of ROS and effectively inhibited the MC-LR-induced hepatocyte apoptosis and liver injury, suggesting that ROS played a critical role in MC-LR-induced hepatocyte apoptosis and liver injury. The protective effect of vitamin C and E also suggested the potential interest in the clinical treatment of MC-LR-induced liver injury and hepatotoxicity.

Transforming growth factor-beta (TGFbeta)-activated signalling pathways can lead to apoptosis, growth arrest or promotion of malignant behaviour, dependent on cellular context. The molecular mechanisms involved in TGFbeta-induced apoptosis remain controversial; although changes in gene expression are thought to be pivotal to the process, several different candidate apoptotic initiators and mediators have been proposed. Smad4, a critical component of the TGFbeta-induced transcriptional machinery, is shown here to be essential for induction of apoptosis. Gene expression analysis identified the proapoptotic Bcl-2 family members, Bmf and Bim, as induced by TGFbeta, dependent on both Smad4 and p38 function and the generation of reactive oxygen species. TGFbeta-induced Bmf and Bim localize to cellular membranes implicated in apoptosis. Inhibition of the TGFbeta-induced expression of both these proteins together provides significant protection of cells from apoptosis. The TGFbeta-triggered cell death programme thus involves induction of multiple BH3-only proteins during the induction of apoptosis.

Two major apoptotic signaling pathways have been defined in mammals, the extrinsic pathway, initiated by ligation of death receptors, and the intrinsic pathway, triggered by cytochrome c release from mitochondria. Here, we identified and characterized the Xenopus homologs of caspase-10 (xCaspase-10beta), a novel initiator caspase, and Bid (xBid), a BH3-only molecule of the Bcl-2 family involved in both the extrinsic and intrinsic pathways. Exogenous expression of these molecules induced apoptosis of mammalian cells. By biochemical and cytological analyses, we clarified that xCaspase-10beta and xBid exhibit structural and functional similarities to their mammalian orthologues. We also detected xCaspase-10beta and xBid transcripts during embryogenesis by whole-mount in situ hybridization and RT-PCR analysis. Microinjection of mRNA encoding a protease-defect xCaspase-10beta mutant into embryos resulted in irregular development. Enforced expression of active xBid induced cell death in developing embryos. Using transgenic frogs established to allow monitoring of caspase activation in vivo, we confirmed that this form of cell death is caspase-dependent apoptosis. Thus, we demonstrated that the machinery governing the extrinsic and intrinsic apoptotic pathways are already established in Xenopus embryos. Additionally, we propose that the functions of the initiator caspase and BH3-only molecule are evolutionarily conserved in vertebrates, functioning during embryonic development.

The function of alpha globin in the context of oxygen transport in erythroid cells is well described. Recently the expression of alpha globin was shown to be upregulated upon specific apoptotic stimuli like cytokine deprivation or cisplatin treatment in the hematopoietic pro-B cell line, FL5.12. In contrast to alpha globin, beta globin or globin-like genes were expressed at a very low level or were not expressed at all. Further, we found that alpha globin was not associated with heme. Apoptotic cells neither produced hemoglobin nor displayed a phenotype of cells differentiating down the erythroid lineage. Also other cell lines of variable differentiation status (NIH3T3, HeLa, K562) upregulated alpha globin during treatment with apoptosis-inducing agents. Under IL-3-deprived conditions GFP-alpha globin accelerated the progression of apoptosis comparable to GFP-Bax. GFP-alpha globin was expressed at a low level and enrichment of FL5.12 cells expressing GFP-alpha globin was difficult even in the presence of IL-3. Caspase-8, -9 and -3 as well as the proapoptotic factor Bax and cytochrome c were activated. Antisense alpha globin downregulated the expression of endogenous alpha globin und reduced caspase activity. Taken together these data indicate that alpha globin is a new and crucial factor in apoptosis especially supporting the mitochondrial pathway.