Effector Caspases Research Articles

The initiator caspase Dronc plays a non-apoptotic role in promoting DNA damage signalling in D. melanogaster.

The phosphorylation of the variant histone H2Ax (denoted γH2Ax; γH2Av in flies) constitutes an important signalling event in DNA damage sensing, ensuring effective repair by recruiting DNA repair machinery. In contrast, the γH2Av response has also been reported in dying cells, where it requires activation of caspase-activated DNases (CADs). Moreover, caspases are known to be required downstream of DNA damage for cell death execution. We show here, for the first time, that the Drosophila initiator caspase Dronc acts as an upstream regulator of the DNA damage response (DDR) independently of executioner caspases by facilitating γH2Av signalling, possibly through a function that is not related to apoptosis. Such a γH2Av response is mediated by ATM rather than ATR, suggesting that Dronc function is required upstream of ATM. In contrast, the role of γH2Av in cell death requires effector caspases and is associated with fragmented nuclei. Our study uncovers a novel function of Dronc in response to DNA damage aimed at promoting DDR via γH2Av signalling in intact nuclei. We propose that Dronc plays a dual role that can either initiate DDR or apoptosis depending upon its level and the required threshold of its activation in damaged cells.This article has an associated First Person interview with the first author of the paper.

The Canonical Inflammasome: A Macromolecular Complex Driving Inflammation.

The inflammasome is a multi-molecular platform crucial to the induction of an inflammatory response to cellular danger. Recognition in the cytoplasm of endogenously and exogenously derived ligands initiates conformational change in sensor proteins, such as NLRP3, that permits the subsequent rapid recruitment of adaptor proteins, like ASC, and the resulting assembly of a large-scale inflammatory signalling platform. The assembly process is driven by sensor-sensor interactions as well as sensor-adaptor and adaptor-adaptor interactions. The resulting complex, which can reach diameters of around 1 micron, has a variable composition and stoichiometry. The inflammasome complex functions as a platform for the proximity induced activation of effector caspases, such as caspase-1 and caspase-8. This ultimately leads to the processing of the inflammatory cytokines pro-IL1β and pro-IL18 into their active forms, along with the cleavage of Gasdermin D, a key activator of cell death via pyroptosis.

Interference with the HSF1/HSP70/BAG3 Pathway Primes Glioma Cells to Matrix Detachment and BH3 Mimetic-Induced Apoptosis.

Malignant gliomas exhibit a high intrinsic resistance against stimuli triggering apoptotic cell death. HSF1 acts as transcription factor upstream of HSP70 and the HSP70 co-chaperone BAG3 that is overexpressed in glioblastoma. To specifically target this resistance mechanism, we applied the selective HSF1 inhibitor KRIBB11 and the HSP70/BAG3 interaction inhibitor YM-1 in combination with the pan-Bcl-2 inhibitor AT-101. Here, we demonstrate that lentiviral BAG3 silencing significantly enhances AT-101-induced cell death and reactivates effector caspase-mediated apoptosis in U251 glioma cells with high BAG3 expression, whereas these sensitizing effects were less pronounced in U343 cells expressing lower BAG3 levels. KRIBB11 decreased protein levels of HSP70, BAG3, and the antiapoptotic Bcl-2 protein Mcl-1, and both KRIBB11 and YM-1 elicited significantly increased mitochondrial dysfunction, effector caspase activity, and apoptotic cell death after combined treatment with AT-101 and ABT-737. Depletion of BAG3 also led to a pronounced loss of cell-matrix adhesion, FAK phosphorylation, and in vivo tumor growth in an orthotopic mouse glioma model. Furthermore, it reduced the plating efficiency of U251 cells in three-dimensional clonogenic assays and limited clonogenic survival after short-term treatment with AT-101. Collectively, our data suggest that the HSF1/HSP70/BAG3 pathway plays a pivotal role for overexpression of prosurvival Bcl-2 proteins and cell death resistance of glioma. They also support the hypothesis that interference with BAG3 function is an effective novel approach to prime glioma cells to anoikis. Mol Cancer Ther; 16(1); 156-68. ©2016 AACR.

Development of a Novel Experimental In Vitro Model of Isothiocyanate-induced Apoptosis in Human Malignant Melanoma Cells.

Isothiocyanates are constituents of cruciferous vegetables which have been associated with reduced cancer risk partially through their ability to induce apoptosis in malignant cells including melanoma. We have utilized human malignant melanoma (A375), epidermoid carcinoma (A431) and immortalized keratinocyte (HaCaT) cells exposed to various isothiocyanates, under different experimental conditions. An experimental in vitro model utilizing low isothiocyanate concentrations (0.1-5 μM for 48 h with all treatments being refreshed after 24h) was shown to be (i) most efficient in exerting an anti-cancer effect when compared to higher concentrations (5-100 μM for 24 or 48 h added as a single bolus) and (ii) specific to A375 cells while A431 and HaCaT cells remained unaffected. Such effect involved the activation of several caspases including (iii) initiator caspases 8, 9, 4 (indicating the involvement of intrinsic, extrinsic and endoplasmic reticulum-based pathways) and (iv) effector caspases 3, 7 and 6. Utilization of low isothiocyanate concentrations (under the conditions described herein) exerts an anti-cancer effect specific to human malignant melanoma cells thus providing a therapeutic basis for their utilization in management of the disease.

Regulation of Linear Ubiquitin Chain Assembly Complex by Caspase-Mediated Cleavage of RNF31.

Cell death and survival signaling pathways have opposed but fundamental functions for various cellular processes and maintain cell homeostasis through cross talk. Here we report a novel mechanism of interaction between these two pathways through the cleavage of RNF31 by caspases. RNF31, a component of the linear ubiquitin chain assembly complex (LUBAC), regulates cell survival by inducing linear ubiquitination of NF-κB signaling components. We found that RNF31 is cleaved under apoptosis conditions through various stimulations. The effector caspases caspase 3 and caspase 6 are responsible for this event, and aspartates 348, 387, and 390 were identified as target sites for this cleavage. Cleavage of RNF31 suppressed its ability to activate NF-κB signaling; thus, mutation of cleavage sites inhibited the induction of apoptosis by treatment with tumor necrosis factor alpha (TNF-α). Our findings elucidate a novel regulatory loop between cell death and the survival signal and may provide guidance for the development of therapeutic strategies for cancers through the sensitization of tumor cells to death-inducing drugs.

Synthesis of acyl oleanolic acid-uracil conjugates and their anti-tumor activity.

BackgroundOleanolic acid, which can be isolated from many foods and medicinal plants, has been reported to possess diverse biological activities. It has been found that the acylation of the hydroxyl groups of the A-ring in the triterpene skeleton of oleanolic acid could be favorable for biological activities. The pyrimidinyl group has been constructed in many new compounds in various anti-tumor studies.ResultsFive acyl oleanolic acid-uracil conjugates were synthesized. Most of the IC50 values of these conjugates were lower than 10.0 μM, and some of them were even under 0.1 μM. Cytotoxicity selectivity detection revealed that conjugate 4c exhibited low cytotoxicity towards the normal human liver cell line HL-7702. Further studies revealed that 4c clearly possessed apoptosis inducing effects, could arrest the Hep-G2 cell line in the G1 phase, induce late-stage apoptosis, and activate effector caspase-3/9 to trigger apoptosis.ConclusionsConjugates of five different acyl OA derivatives with uracil were synthesized and identified as possessing high selectivity toward tumor cell lines. These conjugates could induce apoptosis in Hep-G2 cells by triggering caspase-3/9 activity.Graphical abstractFive acyl oleanolic aicd-uracil conjugates were synthesized. These conjugates exhibited selective cytotoxicity toward tumor cells achieved via inducing apoptosis by activation of caspase-3/9. Electronic supplementary materialThe online version of this article (doi:10.1186/s13065-016-0217-5) contains supplementary material, which is available to authorized users.

Antiapoptotic Effect of Acetylcholine in Fas-Induced Apoptosis in Human Keratocytes.

To investigate the possible antiapoptotic effect of acetylcholine (ACh) in Fas-mediated apoptosis of primary human keratocytes in vitro, and to explore the underlying mechanism. Primary human keratocytes were isolated from healthy corneas. Fas ligand (FasL) was used to induce apoptosis in keratocytes. Cell death was assessed by ELISA. Activity of caspase-3, -7, -8, and -9 was measured with luminescent caspase activity assays. Expression of nuclear factor-κB (NF-κB) gene was assessed with RT-quantitative (q)PCR. Cytochrome c release apoptosis assay kit was used to extract mitochondria and cytosol. Cytochrome c release, cleavage of Bid, and expression of B-cell lymphoma 2 (Bcl-2) were determined by Western blot. Cell death ELISA revealed that ACh is able to reduce Fas-induced apoptosis in keratocytes. Analysis of the activity of effector caspases-3 and -7 showed that ACh, when added to Fas-treated cells, decreases the activation of both these enzymes. The activity of initiator caspases -8 and -9 also decreased when ACh was added to Fas-treated cells. This antiapoptotic effect of ACh was dependent on ACh concentration and activation of muscarinic ACh receptors. Analysis of the antiapoptotic mechanisms triggered by ACh showed that ACh downregulates expression of FasL-induced NF-κB RNA expression, upregulates expression of antiapoptotic protein Bcl-2, downregulates expression of proapoptotic protein Bad, reduces cytochrome c release, and prevents proapoptotic Bid protein cleavage. Acetylcholine has an antiapoptotic effect in a Fas-apoptosis model of human primary keratocytes in vitro. It is therefore possible that ACh may play a role in corneal wound healing, by modulating its initiation phase.

꼬시래기 산추출물의 primary 인체 전립선 암세포 증식억제 효과

본 연구에서는 꼬시래기 산추출물(acid extraction of Gracilaria verrucosa, AEG)을 이용하여 RC-58T/h/SA#4 primary 인체 전립선 암세포에 대한 증식억제 및 apoptosis 유도효과를 밝히고자 하였다. AEG의 처리는 전립선 암세포에서 24시간에서 농도 의존적으로 증식 억제능을 보이는 반면 정상세포에서는 독성을 나타내지 않아 암세포의 증식만을 선택적으로 억제시킴을 확인할 수 있었다. 또한 RC-58T/h/SA#4 세포에서 AEG의 처리는 apoptotic body 형성 및 핵의 형태 변화를 유도하였으며, anti-apoptotic 인자인 Bcl-2 단백질은 감소시키고 pro-apoptotic 인자인 Bax 단백질은 증가시키는 것으로 나타났다. Apoptosis의 유발과 관련된 주요인자인 caspase-3 단백질의 발현은 대조구와 비교하여 AEG를 처리한 군에서 caspase-3의 발현을 농도 의존적으로 증가시키는 것으로 나타났다. 한편, bisphenol A에 의해 비정상적으로 증식된 전립선 암세포에서 AEG의 처리는 유의적인 전립선암세포 성장억제효능을 나타내었다. 본 연구에서는 AEG가 RC-58T/h/SA#4 전립선암 세포에서 암세포 성장억제효과 및 apoptosis 유도효과를 나타낸다는 것을 확인하였으며, 환경호르몬에 의해 증식된 암에 대해서도 성장을 억제할 수 있는 효능을 가지고 있음을 증명하였다. The purpose of this study was to investigate the anti-proliferative and apoptotic effects of acid extract of Gracilaria verrucosa (AEG) on RC-58T/h/SA#4 primary human prostate cancer cells. AEG significantly decreased the cell viability of prostate cancer cells in a dose-dependent manner. AEG also showed relatively low cytotoxicity on normal cell (RWPE-1). The morphology of prostate cancer cells treated with AEG was distorted to shrunken cell masses. In addition, it was revealed that AEG induced cell death as evidenced by increased formation of apoptotic body and nuclear condensation. Furthermore, AEG clearly modulated the down regulation of Bcl-2 (anti-apoptotic)/Bax (pro-apoptotic) family and activated caspase-3 as an effector caspase in a dose-dependent manner. AEG inhibited cell proliferation induced by environmental hormones as a bisphenol A in a dose-dependent manner. These results indicate that AEG act as anti-proliferative effects as a potential therapeutic agent on primary human prostate cancer cells.

Glycidamide inhibits progesterone production through reactive oxygen species-induced apoptosis in R2C Rat Leydig Cells

The food contaminant acrylamide (AA) is usually recognized as a probable human carcinogen. In addition, AA has also been found able to induce male infertility in animals. Interestingly, resent research work revealed that the toxic effect of AA on the ability of male reproduction in vivo may due to glycidamide (GA) which is the metabolite of AA. In this study, R2C Leydig cells was used to investigate the toxic effects of GA on progesterone production. GA caused dose-dependent inhibition on the cell growth, with IC25, IC50, and IC75 values found at 0.635, 0.872, and 1.198 mM, respectively. The results of single cell gel/Comet assay showed that GA significantly induced early-phase cell apoptosis, reduced progesterone production, as well as decreasing the protein expression of steroidogenic acute regulatory (StAR) in R2C cells. Furthermore, GA induced overproduction of intracellular reactive oxygen species (ROS), upregulated Bax expression, decreased mitochondrial membrane potential, and triggered mitochondria-mediated cell apoptosis. Consequently, the downstream effector caspase-3 was activated, resulting in Leydig cells apoptosis. Overall, our results showed that GA could damage R2C Leydig cells by the lesion of the ability of progesterone genesis and inducing cells apoptosis.

Oncolytic vaccinia virus combined with radiotherapy induces apoptotic cell death in sarcoma cells by down-regulating the inhibitors of apoptosis.

Advanced extremity melanoma and sarcoma present a significant therapeutic challenge, requiring multimodality therapy to treat or even palliate disease. These aggressive tumours are relatively chemo-resistant, therefore new treatment approaches are urgently required. We have previously reported on the efficacy of oncolytic virotherapy (OV) delivered by isolated limb perfusion. In this report, we have improved therapeutic outcomes by combining OV with radiotherapy. In vitro, the combination of oncolytic vaccinia virus (GLV-1h68) and radiotherapy demonstrated synergistic cytotoxicity. This effect was not due to increased viral replication, but mediated through induction of intrinsic apoptosis. GLV-1h68 therapy downregulated the anti-apoptotic BCL-2 proteins (MCL-1 and BCL-XL) and the downstream inhibitors of apoptosis, resulting in cleavage of effector caspases 3 and 7. In an in vivo ILP model, the combination of OV and radiotherapy significantly delayed tumour growth and prolonged survival compared to single agent therapy. These data suggest that the virally-mediated down-regulation of anti-apoptotic proteins may increase the sensitivity of tumour cells to the cytotoxic effects of ionizing radiation. Oncolytic virotherapy represents an exciting candidate for clinical development when delivered by ILP. Its ability to overcome anti-apoptotic signals within tumour cells points the way to further development in combination with conventional anti-cancer therapies.

Parallel single-cell analysis of active caspase-3/7 in apoptotic and non-apoptotic cells

Analysing thechemical content of individual cells has already been proven to reveal unique information on various biological processes. Single-cell analysis provides more accurate and reliable results for biology and medicine than analyses of extracts from cell populations, where a natural heterogeneity is averaged. To meet the requirements in the research of important biologically active molecules, such as caspases, we have developed a miniaturized device for simultaneous analyses of individual cells. A stainless steel body with a carousel holder enables high-sensitivity parallel detections in eight microvials. The holder is mounted in front of a photomultiplier tube with cooled photocathode working in photon counting mode. The detection of active caspase-3/7, central effector caspases in apoptosis, in single cells is based on the bioluminescence chemistry commercially available as Caspase-Glo® 3/7 reagent developed by Promega. Individual cells were captured from a culture medium under microscope and transferred by micromanipulator into detection microvial filled with the reagent. As a result of testing, the limits of detection and quantification were determined to be 0.27/0.86 of active caspase-3/7 content in an average apoptotic cell and 0.46/2.92 for non-apoptotic cells. Application potential of this technology in laboratory diagnostics and related medical research is discussed. Graphical abstract Miniaturized device for simultaneous analyses of individual cells.

Caspase-1 from the silkworm, Bombyx mori, is involved in Bombyx mori nucleopolyhedrovirus infection.

Caspase-1 is one of the effector caspases in mammals that plays a central role in apoptosis. However, the lepidopteran caspase-1, especially the Bombyx mori caspase-1 (Bm-caspase-1), has not been investigated in detail. In this study, Bm-caspase-1 was identified from an expressed sequence tag database in B. mori by BLAST search. The open reading frame of Bm-caspase-1 contained 879 nucleotides and encoded 293 amino acids with a predicted molecular mass of 33 kDa. Bm-caspase-1 contained two consensus amino acid motifs of caspase cleavage sites, DEGDA and TETDG. Caspase activity assays revealed significant proteolytic activity of the Ac-DEVD-pNA substrate. Bm-caspase-1 can be detected in all tissues and developmental stages by a semi quantitative polymerase chain reaction assay. More importantly, the expression level of Bm-caspase-1 is increased upon baculovirus infection and up-regulated in BmNPV-resistant silkworms. Taken together, these results indicate that Bm-caspase-1 plays an important role during baculovirus infection.

Possible mechanisms of Leukoagglutinin induced apoptosis in human cells in vitro.

Leukoagglutinin is one of the phytohemagglutinin isolectins isolated from Phaseolus vulgaris. In our recent study, we showed that this lectin is able to influence the growth of human cancer cells in vitro. In addition, using the acridine orange and ethidium bromide staining, we found that leukoagglutinin can induce apoptosis. In order to understand the molecular mechanisms of induction of apoptosis, we performed computational modeling with subsequent experimental verification of theoretical data in vitro. We developed computational models of leukoagglutinin interaction with pro- (FasR and TNFR) and anti-apoptotic (IGF-1 and EGFR) receptors, and confirmed that leukoagglutinin may specifically interact with these receptors. Furthermore, we proved that leukoagglutinin can induce apoptosis in cancer (HEp-2) and non-cancer (4BL) cells, and observed that PHA-L is able to induce apoptosis through the up-regulation of Bax protein and activation of the effector caspase-3 and initiator caspase-8. However, these proteins have no effect on the Bcl-2 expression level.

Silver nanoparticles induce irremediable endoplasmic reticulum stress leading to unfolded protein response dependent apoptosis in breast cancer cells.

Nowadays, silver nanoparticles (AgNP) are widely used in the medical field mainly for their antibacterial properties. Although some studies report a cytotoxic activity of the particles, the mechanisms involved in AgNP-induced cell death remain to be determined. Herein, we report that AgNP of 2 (AgNP2) and 15nm (AgNP15) induce apoptosis in human MCF-7 and T-47D breast cancer cells. Treatment with AgNP2 and AgNP15 led to accumulation and aggregation of misfolded proteins causing an endoplasmic reticulum (ER) stress and activating the unfolded protein response (UPR). The three main ER sensors, PERK, IRE-1α and ATF-6, were rapidly activated in response to AgNP2 and AgNP15. Although Grp78 levels remained unchanged, AgNP2 and AgNP15 induced upregulation of the transcription factors ATF-4 and GADD153/CHOP. Moreover, the initiating caspase-9 and the effector caspase-7 were activated in response to these NPs. The expression levels of the pro-apoptotic BIM and BAD proteins remained unchanged. In contrast, a downregulation of Mcl-1 and xIAP protein expression as well as a processing of PARP were observed. Pharmacological inhibition of PERK kinase and IRE-1 endonuclease activities, as well as inhibition of ER-stress, partially protected cells from AgNP2- and AgNP15-induced apoptosis. Of note, the non-cancerous MCF-10A cells were more resistant to both AgNP2 and AgNP15 when compared to MCF-7 and T-47D cell lines. Taken together, our results demonstrate that AgNP induce ER stress and can target the UPR-dependent apoptotic pathway in MCF-7 and T-47D, which highlights new potential strategies for the treatment of breast cancers.

Investigation of cellular mechanisms involved in apoptosis induced by a synthetic naphthylchalcone in acute leukemia cell lines.

We have previously reported the cytotoxic effects of chalcone A1, derived from 1-naphthaldehyde, in leukemia cell lines. On the basis of these findings, the main aim of this study was to elucidate some of the molecular mechanisms involved in apoptosis induced by chalcone A1 toward K562 and Jurkat cells. In both cell lines, chalcone A1 decreased the mitochondrial membrane potential, increased the expression of Bax proapoptotic protein, and decreased the expression of Bcl-2 antiapoptotic protein (resulting in the inversion of the Bcl-2/Bax ratio), which indicates the involvement of the intrinsic pathway. In addition, chalcone A1 increased the expression of FasR in Jurkat cells, which also indicates the involvement of the extrinsic pathway in this cell line. The results also showed an increased expression of effector caspase-3 and cleaved PARP-1 and a decreased expression of IAP protein survivin, which are consistent with apoptotic cell death. The decreased expression of Ki67 suggests that the mechanism involved in cell death induced by chalcone A1 also involves a decrease in cell proliferation. In ex-vivo experiments, chalcone A1 reduced the cell viability of blast cells collected from eight patients with different types of acute leukemia, confirming the cytotoxicity results found in vitro. The results obtained so far are very promising and further studies need to be carried out so that chalcone A1 can be used as a prototype for the development of new antileukemia agents.

A549 폐암세포에서 미토콘드리아 경로를 통한 개똥쑥 추출물의 apoptosis 유도 효과

개똥쑥 추출물(AAE)은 암에 효과적인 약초로 알려져 있다. Apoptosis는 프로그램화된 세포사멸로 미토콘드리아는 세포사멸 조절에 중요한 역할을 한다. 이 연구는 A549 폐암세포에서 Bcl-2 하위조절과 미토콘드리아 경로를 통한 AAE의 p53 비의존적인 세포사멸을 보여주고 있다. AAE는 p-Akt, cox-2, p53 그리고 미토콘드리아 조절 단백질을 통해 암세포의 사멸을 촉진한다. p-Akt/cox-2 단백질은 세포 증식과 생존에 중요한 역할을 한다고 알려져 있다. Bax, Bak, Bim과 같이 세포사멸을 촉진하는 Bcl-2 단백질은 미토콘드리아 외막의 투과성을 조절한다. AAE의 처리는 p-Akt, p-Mdm2, cox-2 그리고 anti-apoptotic 단백질과 같이 세포사멸을 억제하는 단백질들의 발현을 감소시키는 반면에 암 억제자인 p53과 pro-apoptotic 단백질들을 증가시킨다. Bax/Bak의 활성화는 caspase를 활성화시키기 위해 cytochrome c를 미토콘드리아에서 세포질로 방출하도록 한다. Caspase-3는 apoptosis 과정과 관련된 주요 effector caspase이다. Caspase-3는 일반적으로 pro-enzyme형태로 세포질에 존재한다. Apoptosis의 개시단계에서 caspase-3는 proteolytic cleavage에 의해 활성화되고 활성화된 caspase-3는 PARP를 분해한다. Apoptosis와 관련된 단백질들의 신호전달 사이의 상관관계를 알기 위해 Pifithrin-α (p53 inhibitor)와 Celecoxib (cox-2 inhibitor)을 처리했다. 이러한 결과를 통해 A549 폐암 세포에 AAE를 처리하였을 때 p53-independent 경로를 통해 apoptosis가 유도된다는 것을 확인하였다. The extract from Artemisia annuain L.(AAE) is known as a medicinal herb that is effective against cancer. Apoptosis is the process of programmed cell death, and mitochondria are known to play a central role in cell death control. In this study, we evaluated the p53-independent apoptosis of extract of AAE through downregulation of Bcl-2 and the mitochondrial pathway in A549 (lung cancer cells). AAE may exert cancer cell apoptosis through regulating p-Akt, Cox-2, p53 and mitochondria-mediated apoptotic proteins. p-Akt/cox-2 is known to play an important role in cell proliferation and cell survival. The Bcl-2 pro-apoptotic proteins (such as Bax, Bak and Bim) mediate the permeabilization of the mitochondrial outer membrane. Treatment of AAE reduces p-Akt, p-Mdm2, cox-2 and anti-apoptotic proteins (such as Bcl-2), while tumor suppressor p53 and pro-apoptotic proteins. Activation of Bax/Bak releases cytochrome c from mitochondria to the cytosol to activate a caspase. Caspase-3 is the major effector caspase associated with apoptotic pathways. Caspase-3 generally exists in cytoplasm in the form of a pro-enzyme. In the initiation stage of apoptosis, caspase-3 is activated by proteolytic cleavage and activated caspase-3 cleaves poly (ADP-ribose) polymerase (PARP). We treated Pifithrin-α (p53 inhibitor) and Celecoxib (Cox-2 inhibitor) to learn the relationship between the signal transduction of proteins associated with apoptosis. These results suggest that AAE induces apoptosis through a p53-independent pathway in A549.

Bakkenolide A inhibits leukemia by regulation of HDAC3 and PI3K/Akt-related signaling pathways.

Leukemia has been the third type of cancer killing many people across the world. Bakkenolide A (Bak), extracted from Petasites tricholobus, has been suggested to against cancer and display protective effects on inflammatory cytokines formation. And increasing evidences suggest that histone deacetylase 3 (HDAC3) plays vital roles in cancer formation and persistence via cell death, apoptosis and inflammation. But the function of Bakkenolide A in regulating leukemia is not understood yet, particularly via HDAC3. Here, we found that HDAC3 is up-regulated in clinical samples of leukemia compared with adjacent normal tissues. Then the expression of HDAC3 was knocked down via RNA interference in K562 cells. And inhibition of HDAC3 expression is able to improve leukemia invasion, migration and proliferation. Further, we also found HDAC3 bound to IκBα, affecting subsequent inflammation response. Moreover, Bakkenolide A was found to inhibit inflammation, induce apoptosis and cell death in leukemia cells via PI3K-regulated signaling pathway, down-regulating IKKs expression and suppressing in proinflammatory cytokines of IL-1β, IL-18 and TNF-α. Up-regulation of Caspase3/7 was observed in cells of HDAC3-knockdown and Bakkenolide A treatment, inducing leukemia cell apoptosis. Also, the expression of Akt and GSK were activated by HDAC3-knockdown and Bakkenolide A-treatment. Thus, these results indicated that Bakkenolide A-mediated HDAC3 sensitization in leukemia cells seem to be associated with activation of effector IKKs, Akt/GSK, and caspases through induction of the PI3K pathway, leading to inflammation, cell death, and apoptosis.

Identification of AaCASPS7, an effector caspase in Aedes albopictus

Aedes albopictus mosquito is a vector of various arboviruses and is becoming a significant threat to public health due to its rapid global expansion. Several reports suggest that apoptosis could be a factor limiting arbovirus infection in mosquitoes. Thus, it is significant to identify apoptosis pathway and study the correlation between apoptosis and virus infection in mosquitoes. Apoptosis is a type of programmed cell death that plays a vital role in immunity, development, and tissue homeostasis. Caspases are a family of conserved proteases playing important roles in apoptosis. In this study, we identified Aedes albopictus AaCASPS7, a caspase shared high identity with dipteran insect drICE orthologs. Phylogenetic analysis showed the closest relative of AaCASPS7 was Aedes aegypti AeCASPS7. AaCASPS7 displayed several features that were typical of an effector caspase and showed significant activity to effector caspase substrates. Aacasps7 transcripts were expressed ubiquitously in developmental and adult stages in Aedes albopictus mosquitoes. Transient expression of AaCASPS7 induced caspase-dependent apoptosis in C6/36 cells. Taken together the above data, this study identified a novel caspase, AaCASPS7, which might function as an apoptotic caspase. Further study the function of AaCASPS7 would facilitate better understanding the apoptotic mechanism in Aedes albopictus mosquito.

Autophagy confers resistance to lipopolysaccharide-induced mouse hepatocyte injury.

During sepsis, bacterial products, particularly LPS, trigger injury in organs such as the liver. This common condition remains largely untreatable, in part due to a lack of understanding of how high concentrations of LPS cause cellular injury. In the liver, the lysosomal degradative pathway of autophagy performs essential hepatoprotective functions and is induced by LPS. We, therefore, examined whether hepatocyte autophagy protects against liver injury from septic levels of LPS. Mice with an inducible hepatocyte-specific knockout of the critical autophagy gene Atg7 were examined for their sensitivity to high-dose LPS. Increased liver injury occurred in knockout mice, as determined by significantly increased serum alanine aminotransferase levels, histological evidence of liver injury, terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling, and effector caspase-3 and -7 activation. Hepatic inflammation and proinflammatory cytokine induction were unaffected by the decrease in hepatocyte autophagy. Although knockout mice had normal NF-κB signaling, hepatic levels of Akt1 and Akt2 phosphorylation in response to LPS were decreased. Cultured hepatocytes from knockout mice displayed a generalized defect in Akt signaling in response to multiple stimuli, including LPS, TNF, and IL-1β. Akt activation mediates hepatocyte resistance to TNF cytotoxicity, and anti-TNF antibodies significantly decreased LPS-induced liver injury in knockout mice, indicating that the loss of autophagy sensitized to TNF-dependent liver damage. Hepatocyte autophagy, therefore, protects against LPS-induced liver injury. Conditions such as aging and steatosis that impair hepatic autophagy may predispose to poor outcomes from sepsis through this mechanism.

Abstract 1287: The dual IAP antagonist, ASTX660, increases the anti-tumor activity of paclitaxel in preclinical models of triple-negative breast cancer in vivo

Abstract Background: Paclitaxel-mediated secretion of inflammatory mediators, including TNFα, potentially creates paracrine and autocrine signaling loops that can reduce paclitaxel-induced apoptosis with resultant cancer cell survival (paclitaxel resistance). One mechanism of cancer cell survival is the expression of inhibitor of apoptosis proteins (IAPs): Cellular IAP (cIAP) is involved in inflammatory pro-survival NF-κB activation, blocking the activation of effector caspases 3 and 7, while X-linked IAP (XIAP) directly binds the effector caspases 3, 7 and 9, inhibiting the full activation of the apoptotic pathway. Antagonism of IAPs in the setting of paclitaxel treatment may enhance the cancer cell death by switching the chemotherapy-induced inflammatory TNFα signaling from pro-survival to apoptosis. ASTX660 is an orally bioavailable dual antagonist of cIAP and XIAP, currently being investigated in a single-agent Phase 1/2 clinical trial in patients with advanced solid tumors and lymphomas (NCT02503423). Here, we characterize the activity of ASTX660 in triple-negative breast cancer (TNBC) preclinical models as a single agent and in combination with paclitaxel. Results: We first investigated the viability of 21 TNBC cell lines treated with ASTX660 in vitro and found that 43% were sensitive either to ASTX660 alone (MDA-MB-231 and HCC38) or in the presence of exogenous TNFα (HCC1806, Hs578T, BT549, HCC1395, DU4475, MDA-MB-453 and mouse EMT6). In HCC1806 xenografts in mice, both ASTX660 (daily oral treatment) and paclitaxel (weekly intravenous treatment) as single agents caused moderate tumor growth inhibition but not regression. ASTX660 and paclitaxel combination treatment, however, caused regression and all tumors achieved a partial response by Day 19 of treatment. An increase in circulating acute-phase-proteins in animals was also observed on paclitaxel treatment. Conclusions: Our study suggests that paclitaxel treatment causes an inflammatory response which could qualitatively change the tumor environment. Inhibition of IAPs in such inflammatory environment induced by paclitaxel may lead to rapid apoptotic cell death. Thus, addition of IAP antagonist may be a promising approach to enhance the TNBC response to paclitaxel therapy. Citation Format: Tomoko Smyth, Yoko Nakatsuru, Ryoto Fujita, George Ward, Luke Bevan, Jon Lewis, Nicola Wallis, John Lyons. The dual IAP antagonist, ASTX660, increases the anti-tumor activity of paclitaxel in preclinical models of triple-negative breast cancer in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1287.