Death Stimuli Research Articles

Orchestration of Force Generation and Nuclear Collapse in Apoptotic Cells.

Apoptosis, or programmed cell death, is a form of cell suicide that is extremely important for ridding the body of cells that are no longer required, to protect the body against hazardous cells, such as cancerous ones, and to promote tissue morphogenesis during animal development. Upon reception of a death stimulus, the doomed cell activates biochemical pathways that eventually converge on the activation of dedicated enzymes, caspases. Numerous pieces of information on the biochemical control of the process have been gathered, from the successive events of caspase activation to the identification of their targets, such as lamins, which constitute the nuclear skeleton. Yet, evidence from multiple systems now shows that apoptosis is also a mechanical process, which may even ultimately impinge on the morphogenesis of the surrounding tissues. This mechanical role relies on dramatic actomyosin cytoskeleton remodelling, and on its coupling with the nucleus before nucleus fragmentation. Here, we provide an overview of apoptosis before describing how apoptotic forces could combine with selective caspase-dependent proteolysis to orchestrate nucleus destruction.

Retraction of "Breast Cancer Chemo-immunotherapy through Liposomal Delivery of an Immunogenic Cell Death Stimulus Plus Interference in the IDO-1 Pathway".

RETURN TO ISSUEPREVRetractionNEXTORIGINAL ARTICLEThis notice is a retraction.Retraction of “Breast Cancer Chemo-immunotherapy through Liposomal Delivery of an Immunogenic Cell Death Stimulus Plus Interference in the IDO-1 Pathway”Jianqin LuJianqin LuMore by Jianqin Lu, Xiangsheng LiuXiangsheng LiuMore by Xiangsheng Liu, Yu-Pei LiaoYu-Pei LiaoMore by Yu-Pei Liao, Xiang WangXiang WangMore by Xiang Wang, Ayman AhmedAyman AhmedMore by Ayman Ahmed, Wen JiangWen JiangMore by Wen Jiang, Ying JiYing JiMore by Ying Ji, Huan MengHuan MengMore by Huan Meng, and Andre E. NelAndre E. NelMore by Andre E. NelCite this: ACS Nano 2021, 15, 6, 10735Publication Date (Web):May 21, 2021Publication History Received4 May 2021Published online21 May 2021Published inissue 22 June 2021https://doi.org/10.1021/acsnano.0c08653Copyright © 2021 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views2303Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (528 KB) Get e-Alerts Get e-Alerts

Glutathione-Depleting Nanomedicines for Synergistic Cancer Therapy.

Cancer cells frequently exhibit resistance to various molecular and nanoscale drugs, which inevitably affects the drugs' therapeutic outcomes. Overexpression of glutathione (GSH) has been observed in many cancer cells, and solid evidence has corroborated the resulting tumor resistance to a variety of anticancer therapies, suggesting that this biochemical characteristic of cancer cells can be developed as a potential target for cancer treatments. The single treatment of GSH-depleting agents can potentiate the responses of the cancer cells to different cell death stimuli; therefore, as an adjunctive strategy, GSH depletion is usually combined with mainstream cancer therapies for enhancing the therapeutic outcomes. Propelled by the rapid development of nanotechnology, GSH-depleting agents can be readily constructed into anticancer nanomedicines, which have shown a steep rise over the past decade. Here, we review the common GSH-depleting nanomedicines which have been widely applied in synergistic cancer treatments in recent years. Some current challenges and future perspectives for GSH depletion-based cancer therapies are also presented. With the understanding of the structure-property relationship and action mechanisms of these biomaterials, we hope that the GSH-depleting nanotechnology will be further developed to realize more effective disease treatments and even achieve successful clinical translations.

Silicasome Nanocarriers: Development of Facile and Versatile Platinum Drug Delivering Silicasome Nanocarriers for Efficient Pancreatic Cancer Chemo‐Immunotherapy (Small 14/2021)

Effective delivery of activated oxaliplatin to pancreatic cancer achieved by a tailored silicasome nanocarrier is reported by Andre E. Nel, Huan Meng, and co-workers in article number 2005993. This provides a strong immunogenic cell death stimulus that turns the “cold” immunological status into “hot” at tumor site, enabling superior chemo-immunotherapy synergy with anti-PD-1.

Conversion of the death inhibitor ARC to a killer activates pancreatic β cell death in diabetes.

Loss of insulin-secreting pancreatic β cells through apoptosis contributes to the progression of type 2 diabetes, but underlying mechanisms remain elusive. Here, we identify a pathway in which the cell death inhibitor ARC paradoxically becomes a killer during diabetes. While cytoplasmic ARC maintains β cell viability and pancreatic architecture, a pool of ARC relocates to the nucleus to induce β cell apoptosis in humans with diabetes and several pathophysiologically distinct mouse models. β cell death results through the coordinate downregulation of serpins (serine protease inhibitors) not previously known to be synthesized and secreted by β cells. Loss of the serpin α1-antitrypsin from the extracellular space unleashes elastase, triggering the disruption of β cell anchorage and subsequent cell death. Administration of α1-antitrypsin to mice with diabetes prevents β cell death and metabolic abnormalities. These data uncover a pathway for β cell loss in type 2 diabetes and identify an FDA-approved drug that may impede progression of this syndrome.

Proposed mechanism for regulation of H2 O2 -induced programmed cell death in plants by binding of cytochrome c to 14-3-3 proteins.

Programmed cell death (PCD) is crucial for development and homeostasis of all multicellular organisms. In human cells, the double role of extra-mitochondrial cytochrome c in triggering apoptosis and inhibiting survival pathways is well reported. In plants, however, the specific role of cytochrome c upon release from the mitochondria remains in part veiled yet death stimuli do trigger cytochrome c translocation as well. Here, we identify an Arabidopsis thaliana 14-3-3ι isoform as a cytosolic cytochrome c target and inhibitor of caspase-like activity. This finding establishes the 14-3-3ι protein as a relevant factor at the onset of plant H2 O2 -induced PCD. The in vivo and in vitro studies herein reported reveal that the interaction between cytochrome c and 14-3-3ι exhibits noticeable similarities with the complex formed by their human orthologues. Further analysis of the heterologous complexes between human and plant cytochrome c with plant 14-3-3ι and human 14-3-3ε isoforms corroborated common features. These results suggest that cytochrome c blocks p14-3-3ι so as to inhibit caspase-like proteases, which in turn promote cell death upon H2 O2 treatment. Besides establishing common biochemical features between human and plant PCD, this work sheds light onto the signaling networks of plant cell death.

EXTH-60. CHARACTERIZATION OF THE ONCOLYTIC ADENOVIRUS DELTA-24-RGD AS THERAPEUTIC AGENT FOR THE TREATMENT OF THE PEDIATRIC EMBRYONAL BRAIN TUMORS AT/RT AND CNS-PNET

Abstract Atypical teratoid/rhabdoid tumors (AT/RTs) and primitive neuroectodermal tumors of the central nervous system (CNS-PNET) are rare pediatric brain tumors affecting mainly infants and young children. Current therapies for these diseases are inefficient and often cause severe side effects; thus, new therapeutic strategies are urgently needed. Here, we evaluate the anti-tumor effect of the Delta-24-RGD oncolytic adenovirus in preclinical models of CNS-PNET and AT/RT. In vitro, the virus infects and efficiently replicates in different cell culture models of AT/RT and CNS-PNET, inducing a dose-dependent cytotoxic effect (IC50 values below 1 PFU/cell) and the release of immunogenic cell death stimuli. Survival studies in mice bearing orthotopic AT/RTs (supra and infratentorial) or CNS-PNETs demonstrate the therapeutic benefit of a single Delta-24-RGD intratumoral administration (107 or 108 PFU). In virus-treated mice, median overall survival increased between 50–300% (depending on the model), obtaining up to 60% of long-term survivors. Intra-ventricular injection of Delta-24-RGD hinders the development of secondary AT/RT spinal lesions. Moreover, response to the treatment was observed even delaying viral administration up to 30 days after tumor engraftment; thus, demonstrating therapeutic benefit even in animals challenged with large well-established tumor masses. The effect of the oncolytic virus was also assessed in an immunocompetent environment using hCD34+ humanized NSG-SGM3 mice. In this model, Delta-24-RGD significantly extended overall survival (from 23 to 24 days in AT/RT model and from 28 to 55 days in CNS-PNET) with no signs of toxicity associated with inflammation. Immunophenotyping of Delta-24-RGD treated tumors by flow cytometry, RNAseq and multiplexed histology revealed that Delta-24-RGD contributes to generate a pro-inflammatory status, allowing the infiltration of immune cells and the trigger of an antitumor immune response. In conclusion, these results demonstrate that Delta-24-RGD could be a feasible therapeutic choice for AT/RT and CNS-PNET, paving the way for upcoming clinical trials.

Reactivating latent HIV with PKC agonists induces resistance to apoptosis and is associated with phosphorylation and activation of BCL2.

Eradication of HIV-1 by the “kick and kill” strategy requires reactivation of latent virus to cause death of infected cells by either HIV-induced or immune-mediated apoptosis. To date this strategy has been unsuccessful, possibly due to insufficient cell death in reactivated cells to effectively reduce HIV-1 reservoir size. As a possible cause for this cell death resistance, we examined whether leading latency reversal agents (LRAs) affected apoptosis sensitivity of CD4 T cells. Multiple LRAs of different classes inhibited apoptosis in CD4 T cells. Protein kinase C (PKC) agonists bryostatin-1 and prostratin induced phosphorylation and enhanced neutralizing capability of the anti-apoptotic protein BCL2 in a PKC-dependent manner, leading to resistance to apoptosis induced by both intrinsic and extrinsic death stimuli. Furthermore, HIV-1 producing CD4 T cells expressed more BCL2 than uninfected cells, both in vivo and after ex vivo reactivation. Therefore, activation of BCL2 likely contributes to HIV-1 persistence after latency reversal with PKC agonists. The effects of LRAs on apoptosis sensitivity should be considered in designing HIV cure strategies predicated upon the “kick and kill” paradigm.

Do I dislike what you dislike? Investigating the effect of disgust on time processing.

Time perception can be distorted by emotional stimuli. The present study aims to investigate the effect of disgust on time perception in young adults. Here, we report two experiments in which a time bisection task was used with intervals lasting 400ms (short standard) to 1600ms (long standard). In Experiment 1, temporal intervals were marked by neutral images or images from food (rotten, joyful), and facial (disgust, happy) categories. In Experiment 2, disgust-eliciting and neutral stimuli belonging to seven different domains were used: faces, food, animals, body products, injury/infections, death and hygiene. Results showed temporal overestimations when, compared to neutral conditions, disgusted faces (Experiments 1 and 2) and disgusting death and hygiene stimuli (Experiment 2) were used, and a temporal underestimation when images of rotten food (Experiment 1) were used. Results are discussed in terms of arousal-based and attention-based processes and showed that the degree of the emotional component influences time perception.

Liposomal Delivery of Mitoxantrone and a Cholesteryl Indoximod Prodrug Provides Effective Chemo-immunotherapy in Multiple Solid Tumors.

We developed a custom-designed liposome carrier for codelivery of a potent immunogenic cell death (ICD) stimulus plus an inhibitor of the indoleamine 2,3-dioxygenase (IDO-1) pathway to establish a chemo-immunotherapy approach for solid tumors in syngeneic mice. The carrier was constructed by remote import of the anthraquinone chemotherapeutic agent, mitoxantrone (MTO), into the liposomes, which were further endowed with a cholesterol-conjugated indoximod (IND) prodrug in the lipid bilayer. For proof-of-principle testing, we used IV injection of the MTO/IND liposome in a CT26 colon cancer model to demonstrate the generation of a robust immune response, characterized by the appearance of ICD markers (CRT and HMGB-1) as well as evidence of cytotoxic cancer cell death, mediated by perforin and granzyme B. Noteworthy, the cytotoxic effects involved natural killer (NK) cell, which suggests a different type of ICD response. The immunotherapy response was significantly augmented by codelivery of the IND prodrug, which induced additional CRT expression, reduced number of Foxp3+ Treg, and increased perforin release, in addition to extending animal survival beyond the effect of an MTO-only liposome. The outcome reflects the improved pharmacokinetics of MTO delivery to the cancer site by the carrier. In light of the success in the CT26 model, we also assessed the platform efficacy in further breast cancer (EMT6 and 4T1) and renal cancer (RENCA) models, which overexpress IDO-1. Encapsulated MTO delivery was highly effective for inducing chemo-immunotherapy responses, with NK participation, in all tumor models. Moreover, the growth inhibitory effect of MTO was enhanced by IND codelivery in EMT6 and 4T1 tumors. All considered, our data support the use of encapsulated MTO delivery for chemo-immunotherapy, with the possibility to boost the immune response by codelivery of an IDO-1 pathway inhibitor.

Abstract B035: Identification of a tumor cell adaptive stress response signaling pathway, which drives aggressive breast cancer phenotype and therapeutic resistance, in African American patients with locally advanced breast cancer subtypes

Abstract Introduction: The higher incidence of biologically aggressive and treatment-resistant breast cancer (BCa) subtypes, which includes triple negative (TN), basal, and inflammatory breast cancer (IBC), contributes to worse survival outcomes in African American/AA patients, 42% higher death rate compared to White BCa. Amongst locally advanced BCa, IBC is a model of aggressive, most lethal, frequently basal/TN, accounting for 10% AA BCa and disparity in outcomes exist after adjusting for socioeconomic and diagnosis factors. IBC exhibits a unique phenotype wherein malignant cells do not form solid masses and instead, present as hyperproliferative tumor cell clusters, termed tumor emboli, in the skin and lymphatics. Our key finding was the identification of a tumor cell adaptive stress response pathway linking the mitogen activated ser/thr kinase/MNK, X-linked inhibitor of anti-apoptotic protein, XIAP, and nuclear transcription factor, NFkB, mediated proliferative, invasive and immunosuppressive signaling. Our goal was to determine the role of the adaptive stress response pathway, which we have identified to contribute to aggressiveness of TNBC and IBC, in AA breast tumor biology. Methods: Expression of MNK, XIAP and NFkB target gene sets, a 40-gene oxidative stress response that correlates with response to cell death stimuli and a 79-gene IBC signature (identified from comparative analysis of IBC and non-IBC biospecimens) were evaluated in The Cancer Genome Atlas (TCGA) breast cancer datasets (1109 tumor; 113 normal) stratified according to race and molecular subtypes (luminal A, B, Her2, basal). The mean expression for each gene in these signatures were also assessed among different subtypes and race using R. Gene Ontology (GO) analysis was performed using GATHER online tool. XIAP immunohistochemistry (IHC) conducted in BCa samples (n=93) from Duke and MDACC biorepositories. Results: TCGA analysis of the IBC-related signatures identified genes differentially expressed (>2-fold change; Wilcoxon rank sum test, FDR<0.05) among BCa subtypes and between AA vs White within a subtype. Additionally, the oxidative stress response metagene was significantly upregulated in AA basal (p=0.037) whereas there were fewer differences in immune-related genes within AA subtypes suggesting attenuated immune response. IHC, to date, shows high XIAP in high grade, ER-negative and in majority of AA BCa and in IBC samples independent of molecular subtypes and grade. Conclusions: We report a heightened adaptive stress response gene signature and identify genes in the MNK:XIAP:NFkB pathway and IBC-related signatures in AA BCa, suggesting potential biomarkers and drug discovery targets. High XIAP expression in high grade BCa, frequent in AA patients, is of therapeutic relevance as XIAP, the most potent caspase inhibitor, is linked to chemo- and immunotherapy resistance and XIAP inhibitors are in clinical trials. Supported by DOD-Breakthrough-W81XWH-17-1-0297; Duke SOM bridge funds; NCI-1P20-CA202925-01A12. Citation Format: Gayathri R Devi, Muthana Al Abo, Larisa Gearhart-Serna, Joseph Geradts, Shannon J McCall, Savitri Krishnamurthy. Identification of a tumor cell adaptive stress response signaling pathway, which drives aggressive breast cancer phenotype and therapeutic resistance, in African American patients with locally advanced breast cancer subtypes [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B035.

Non-Apoptotic Cell Death Signaling Pathways in Melanoma.

Resisting cell death is a hallmark of cancer. Disturbances in the execution of cell death programs promote carcinogenesis and survival of cancer cells under unfavorable conditions, including exposition to anti-cancer therapies. Specific modalities of regulated cell death (RCD) have been classified based on different criteria, including morphological features, biochemical alterations and immunological consequences. Although melanoma cells are broadly equipped with the anti-apoptotic machinery and recurrent genetic alterations in the components of the RAS/RAF/MEK/ERK signaling markedly contribute to the pro-survival phenotype of melanoma, the roles of autophagy-dependent cell death, necroptosis, ferroptosis, pyroptosis, and parthanatos have recently gained great interest. These signaling cascades are involved in melanoma cell response and resistance to the therapeutics used in the clinic, including inhibitors of BRAFmut and MEK1/2, and immunotherapy. In addition, the relationships between sensitivity to non-apoptotic cell death routes and specific cell phenotypes have been demonstrated, suggesting that plasticity of melanoma cells can be exploited to modulate response of these cells to different cell death stimuli. In this review, the current knowledge on the non-apoptotic cell death signaling pathways in melanoma cell biology and response to anti-cancer drugs has been discussed.

Molecular Basis of 14‐3‐3 Protein Dependent Regulation of Caspase‐2

Among all species, caspase‐2 (C2) is the most evolutionarily conserved caspase required for effective initiation of apoptosis following death stimuli. C2 is activated through dimerization and autoproteolytic cleavage and inhibited through phosphorylation at Ser139 and Ser164, within the linker between the caspase recruitment and p19 domains of the zymogen, followed by association with the adaptor protein 14‐3‐3, which maintains C2 in its immature form procaspase (proC2). However, the mechanism of 14‐3‐3‐dependent inhibition of C2 activation remains unclear. Here, we report the structural characterization of the complex between proC2 and 14‐3‐3 by hydrogen/deuterium mass spectrometry (HDX‐MS) and protein crystallography to determine the molecular basis for 14‐3‐3‐mediated inhibition of C2 activation. Our data reveal that the 14‐3‐3 dimer interacts with proC2 not only through ligand‐binding grooves but also through other regions outside the central channel, thus explaining the isoform‐dependent specificity of 14‐3‐3 protein binding to proC2 and the substantially higher binding affinity of 14‐3‐3 protein to proC2 than to the doubly phosphorylated peptide. The formation of the complex between 14‐3‐3 protein and proC2 does not induce any large conformational change in proC2. Furthermore, 14‐3‐3 protein interacts with and masks both the nuclear localization sequence (NLS) and the C‐terminal region of the p12 domain of proC2 through transient interactions in which both the p19 and p12 domains of proC2 are not firmly docked onto the surface of 14‐3‐3. This masked region of p12 domain is involved in caspase‐2 dimerization. Therefore, 14‐3‐3 protein likely inhibits proC2 activation by blocking its dimerization surface.Support or Funding InformationFundingThis study was funded by the Czech Science Foundation (V.O., grant number 19‐00121S), the Czech Academy of Sciences (RVO:67985823 of the Institute of Physiology), project BIOCEV (CZ.1.05/1.1.00.02.0109 and LQ1604) and MEYS CR (LM2015043 CIISB, Biocev, Biophysical methods, Structural Mass Spectrometry).Crystal structure of the caspase‐2 peptide pS139+pS164 bound to the 14‐3‐3 dimer.Figure 1

14-3-3 protein binding blocks the dimerization interface of caspase-2.

Among all species, caspase-2 (C2) is the most evolutionarily conserved caspase required for effective initiation of apoptosis following death stimuli. C2 is activated through dimerization and autoproteolytic cleavage and inhibited through phosphorylation at Ser139 and Ser164 , within the linker between the caspase recruitment and p19 domains of the zymogen, followed by association with the adaptor protein 14-3-3, which maintains C2 in its immature form procaspase (proC2). However, the mechanism of 14-3-3-dependent inhibition of C2 activation remains unclear. Here, we report the structural characterization of the complex between proC2 and 14-3-3 by hydrogen/deuterium mass spectrometry and protein crystallography to determine the molecular basis for 14-3-3-mediated inhibition of C2 activation. Our data reveal that the 14-3-3 dimer interacts with proC2 not only through ligand-binding grooves but also through other regions outside the central channel, thus explaining the isoform-dependent specificity of 14-3-3 protein binding to proC2 and the substantially higher binding affinity of 14-3-3 protein to proC2 than to the doubly phosphorylated peptide. The formation of the complex between 14-3-3 protein and proC2 does not induce any large conformational change in proC2. Furthermore, 14-3-3 protein interacts with and masks both the nuclear localization sequence and the C-terminal region of the p12 domain of proC2 through transient interactions in which both the p19 and p12 domains of proC2 are not firmly docked onto the surface of 14-3-3. This masked region of p12 domain is involved in C2 dimerization. Therefore, 14-3-3 protein likely inhibits proC2 activation by blocking its dimerization surface. DATABASES: Structural data are available in the Protein Data Bank under the accession numbers 6SAD and 6S9K.

The Bcl-2 Family: Ancient Origins, Conserved Structures, and Divergent Mechanisms.

Intrinsic apoptosis, the response to intracellular cell death stimuli, is regulated by the interplay of the B-cell lymphoma 2 (Bcl-2) family and their membrane interactions. Bcl-2 proteins mediate a number of processes including development, homeostasis, autophagy, and innate and adaptive immune responses and their dysregulation underpins a host of diseases including cancer. The Bcl-2 family is characterized by the presence of conserved sequence motifs called Bcl-2 homology motifs, as well as a transmembrane region, which form the interaction sites and intracellular location mechanism, respectively. Bcl-2 proteins have been recognized in the earliest metazoans including Porifera (sponges), Placozoans, and Cnidarians (e.g., Hydra). A number of viruses have gained Bcl-2 homologs and subvert innate immunity and cellular apoptosis for their replication, but they frequently have very different sequences to their host Bcl-2 analogs. Though most mechanisms of apoptosis initiation converge on activation of caspases that destroy the cell from within, the numerous gene insertions, deletions, and duplications during evolution have led to a divergence in mechanisms of intrinsic apoptosis. Currently, the action of the Bcl-2 family is best understood in vertebrates and nematodes but new insights are emerging from evolutionarily earlier organisms. This review focuses on the mechanisms underpinning the activity of Bcl-2 proteins including their structures and interactions, and how they have changed over the course of evolution.

Functional Compartmentalization of HSP60-Survivin Interaction between Mitochondria and Cytosol in Cancer Cells.

Heat shock protein 60 (HSP60) and survivin reside in both the cytosolic and mitochondrial compartments under physiological conditions. They can form HSP60-survivin complexes through protein–protein interactions. Their expression levels in cancer tissues are positively correlated and higher expression of either protein is associated with poor clinical prognosis. The subcellular location of HSP60-survivin complex in either the cytosol or mitochondria is cell type-dependent, while the biological significance of HSP60-survivin interaction remains elusive. Current knowledge indicates that the function of HSP60 partly rests on where HSP60-survivin interaction takes place. HSP60 has a pro-survival function when binding to survivin in the mitochondria through interacting with other factors such as CCAR2 and p53. In response to cell death signals, mitochondrial survivin functions through preventing procaspase activation. Degradation of cytosolic survivin leads to the loss of mitochondrial membrane potential and aberrant mitosis processes. On the other hand, HSP60 release from mitochondria to cytosol upon death stimuli might exert a pro-death function, either through stabilizing Bax, enhancing procaspase-3 activation, or increasing protein ubiquitination. Combining the knowledge of mitochondrial HSP60-survivin complex function, cytosolic survivin degradation effect, and pro-death function upon mitochondria release of HSP60, a hypothetical scenario for HSP60-survivin shuttling upon death stimuli is proposed.

Apoptotic and Proteolytic Attributes and Metabolomic Changes in Postmortem Muscles from Pigs Subjected to Post-Weaning Transport at Different Seasons

ObjectivesPost-weaning transport of pigs was commonly practiced in the swine industry, however, adversely impact animal growth and well-being due to concurrent stress from weaning and transport. Further, our recent study found that post-weaning transport may have long-term effects on final pork quality attributes in terms of inferior texture and water-holding capacity. Heat shock proteins (HSPs) are anti-apoptotic chaperone proteins, protecting against apoptosis under a variety of cell death stimuli including postmortem muscle conversion process. While a potential role of apoptosis in meat tenderization has been proposed, how early life stress influences apoptotic/proteolytic process and metabolism of postmortem muscles is largely unknown. Thus, the study objective was to evaluate apoptotic and proteolytic attributes and metabolomic changes in postmortem muscles of market weight pigs exposed to early life transport/weaning stress at two seasons.Materials and MethodsTwo repetitions of newly weaned pigs (N = 480) were transported for 12 h in a trailer truck during July 2016 (SUMMER) and April 2017 (SPRING) in north-central Indiana. Upon reaching market weight, 10 animals were randomly chosen from each season and slaughtered in January 2017 and September 2017, respectively. Pairs of longissimus dorsi and psoas major muscles from each carcass were separated at 1d and 7d postmortem. Proteolytic and apoptotic factors including desmin, troponin T, calpain 1, HSP27, and aβ-crystallin were quantified using Western-blot assays, and mitochondria membrane permeability (MMP) was evaluated. Metabolome profiles of 1d samples were analyzed using the GC-TOF-MS/MS platform. Multivariate analyses PCA and PLS-DA were used to determine changes of metabolites. Data were analyzed using PROC MIXED of SAS to compare the traits across season, muscle, and aging effects.ResultsPreviously, SUMMER pigs were reported showing decreased body weight, muscling, and fat deposition, as well as increased shear force and water loss during aging. In the present study, SPRING muscles exhibited increases in calpain 1 autolysis and structural protein degradation, coincided with accelerated apoptosis shown as higher MMP compared to the SUMMER counterparts (P < 0.05). Moreover, PCA and PLS-DA clustering indicated distinct metabolome profiles affected by season and muscle. Seasonal effect mainly altered lipid, glucose, and nitrogen metabolism. A group of 16C to 18C fatty acids were increased in SPRING, probably due to increased lipid anabolism during warm growing/finishing season. Changes of urea, ornithine, aspartic acid, and 5’methylthioadenosine suggested increased amino acid catabolism in SUMMER, corroborating the decreased lean and fat accretion. Seasonal changes of key metabolites related to stress response, including histidine, GABA, and ascorbic acid, suggested increased stress defense in SUMMER pigs, which implied the suppression of apoptotic and proteolytic activities.ConclusionTaken together, SUMMER pigs showed suppressed onset of apoptosis with compromised growth and meat quality, possibly due to alternations in seasonal metabolic response. This may in turn affect the proteolytic potential of early postmortem muscles. Further studies elucidating the involvement of apoptotic process in proteolytic activities in postmortem muscles should be warranted.

Gliotoxin Enhances Autophagic Cell Death via the DAPK1-TAp63 Signaling Pathway in Paclitaxel-Resistant Ovarian Cancer Cells.

Death-associated protein kinase 1 (DAPK1) expression induced by diverse death stimuli mediates apoptotic activity in various cancers, including ovarian cancer. In addition, mutual interaction between the tumor suppressor p53 and DAPK1 influences survival and death in several cancer cell lines. However, the exact role and connection of DAPK1 and p53 family proteins (p53, p63, and p73) in drug-resistant ovarian cancer cells have not been studied previously. In this study, we investigated whether DAPK1 induction by gliotoxin derived from marine fungus regulates the level of transcriptionally active p63 (TAp63) to promote apoptosis in an autophagy-dependent manner. Pre-exposure of paclitaxel-resistant ovarian cancer cells to gliotoxin inhibited the expression of multidrug resistant-associated proteins (MDR1 and MRP1-3), disrupted the mitochondrial membrane potential, and induced caspase-dependent apoptosis through autophagy induction after subsequent treatment with paclitaxel. Gene silencing of DAPK1 prevented TAp63-mediated downregulation of MDR1 and MRP1-3 and autophagic cell death after sequential treatment with gliotoxin and then paclitaxel. However, pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, had no effect on the levels of DAPK1 and TAp63 or on the inhibition of MDR1 and MRP1-3. These results suggest that DAPK1-mediated TAp63 upregulation is one of the critical pathways that induce apoptosis in chemoresistant cancer cells.

Molecular profiling of anastatic cancer cells: potential role of the nuclear export pathway

Anastasis is newly discovered process by which cells recover from late-stage apoptosis upon removal of a death stimulus. Recent reports suggest that cells may recover, even after the initiation of mitochondrial outer-membrane permeabilization (MOMP) and caspase activation. Here, we specifically studied the reversibility of late-stage apoptosis in cervical (HeLa) and breast (MDA-MB-231) cancer cells in relation to the extent of MOMP (limited or widespread). In addition, we explored the molecular factors involved in the anastatic process. The extent of MOMP was assessed using time lapse confocal microscopic imaging, considering mitochondrial cytochrome c-GFP release as a marker for MOMP. Anastatic cells were generated by specifically recovering late-stage apoptotic (annexin V/PI positive) cervical and breast cancer cells. Molecular signaling events involved in death reversal were assessed using LC-MS/MS and qRT-PCR. Targeted chemical inhibition and shRNA-based gene silencing studies were employed to explore the role of the nuclear export pathway in anastasis and increased oncogenicity. Time-lapse imaging of drug-treated Cyt-c-GFP expressing cancer cells revealed cell recovery despite widespread MOMP. A few recovered anastatic cells were noted and these were found to proliferate through a selection-type of survival. They showed increased drug-resistance, migration and invasive potential compared to non-anastatic cancer cells. Network analysis using 49 proteins uniquely expressed in anastatic cells indicated upregulation of nuclear export/import, redox and Ras signaling pathways in both HeLa and MDA-MB-231 anastatic cells, indicating common molecular mechanisms in different cell types. Inhibition of XPO1 significantly reduced the recovery of apoptotic cells and abrogated acquired oncogenic transformation in the anastatic cancer cells. Our study indicates that cancer cells can revert from apoptosis even after the induction of widespread MOMP. We noted a significant role of the nuclear-export pathway in the anastatic process of cancer cells. Inhibition of anastasis through the nuclear export pathway may be a potential therapeutic strategy for targeting drug-resistance, metastasis and recurrence problems during cancer treatment.

Death-Associated Protein Kinase 1 as a Promising Drug Target in Cancer and Alzheimer's Disease.

Background: Death-Associated Protein Kinase 1 (DAPK1) plays an important role in apopto-sis, tumor suppression and neurodegeneration including Alzheimer’s Disease (AD).Objective: This review will describe the diverse roles of DAPK1 in the development of cancer and AD, and the current status of drug development targeting DAPK1-based therapies.Methods: Reports of DAPK1 regulation, function and substrates were analyzed using genetic DAPK1 manipulation and chemical DAPK1 modulators.Results: DAPK1 expression and activity are deregulated in cancer and AD. It is down-regulated and/or inactivated by multiple mechanisms in many human cancers, and elicits a protective effect to counteract numerous death stimuli in cancer, including activation of the master regulator Pin1. Moreover, loss of DAPK1 expression has correlated strongly with tumor recurrence and metastasis, suggesting that lack of sufficient functional DAPK1 might contribute to cancer. In contrast, DAPK1 is highly expressed in the brains of most human AD patients and has been identified as one of the genetic factors affecting suscepti-bility to late-onset AD. The absence of DAPK1 promotes efficient learning and better memory in mice and prevents the development of AD by acting on many key proteins including Pin1 and its downstream tar-gets tau and APP. Recent patents show that DAPK1 modulation might be used to treat both cancer and AD.Conclusion: DAPK1 plays a critical role in diverse physiological processes and importantly, its deregula-tion is implicated in the pathogenesis of either cancer or AD. Therefore, manipulating DAPK1 activity and/or expression may be a promising therapeutic option for cancer or AD.