Delivery of Mixed-Lineage Kinase Domain-Like Protein by Vapor Nanobubble Photoporation Induces Necroptotic-Like Cell Death in Tumor Cells

Modern molecular medicine demands techniques to efficiently deliver molecules directly into mammalian cells. As proteins are the final mediators of most cellular pathways, efficient intracellular protein delivery techniques are highly desired. In this respect, photoporation is a promising recent technique for the delivery of proteins directly into living cells. Here, we show the possibility to deliver a model saccharide (FD70) and a model protein (FITC-BSA) into murine B16 melanoma cells by using the vapor nanobubble photoporation technique with an efficiency of 62% and 38%, respectively. Next, we delivered the mixed-lineage kinase domain-like (MLKL) protein, the most terminal mediator of necroptosis currently known, and caspase-8 and -3 protein, which are important proteins in the initiation and execution of apoptosis. A significant drop in cell viability with 62%, 71% and 64% cell survival for MLKL, caspase-8 and caspase-3, respectively, was observed. Remarkably, maximal cell death induction was already observed within 1 h after protein delivery. Transduction of purified recombinant MLKL by photoporation resulted in rapid cell death characterized by cell swelling and cell membrane rupture, both hallmarks of necroptosis. As necroptosis has been identified as a type of cell death with immunogenic properties, this is of interest to anti-cancer immunotherapy. On the other hand, transduction of purified recombinant active caspase-3 or -8 into the tumor cells resulted in rapid cell death preceded by membrane blebbing, which is typical for apoptosis. Our results suggest that the type of cell death of tumor cells can be controlled by direct transduction of effector proteins that are involved in the executioner phase of apoptosis or necroptosis.

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Positive allosteric modulation of P2X7 promotes apoptotic cell death over lytic cell death responses in macrophages

Cell Death and Disease ◽

10.1038/s41419-019-2110-3 ◽

2019 ◽

Vol 10 (12) ◽

Cited By ~ 7

Author(s):

Stefan Bidula ◽

Kshitija Dhuna ◽

Ray Helliwell ◽

Leanne Stokes

Keyword(s):

Cell Death ◽

Mitochondrial Membrane ◽

Caspase 3 ◽

Allosteric Modulation ◽

Cell Swelling ◽

Dependent Manner ◽

Apoptosis Pathway ◽

Membrane Rupture ◽

Cell Death Pathways ◽

Intrinsic Apoptosis Pathway

AbstractP2X7 is an ATP-gated ion channel that is highly expressed by leukocytes, such as macrophages. Here, P2X7 has been demonstrated to be involved in the regulation of various cell death pathways; including apoptosis, pyroptosis, necrosis, and autophagy. However, cell death induction via P2X7 is complex and is reliant upon the nature of the stimulus, the duration of the stimulus, and the cell type investigated. Previous reports state that high extracellular ATP concentrations promote osmotic lysis, but whether positive allosteric modulation of P2X7 in the presence of lower concentrations of ATP condemns cells to the same fate is unknown. In this study, we compared cell death induced by high ATP concentrations, to cell death induced by compound K, a recently identified and potent positive allosteric modulator of P2X7. Based on our observations, we propose that high ATP concentrations induce early cell swelling, loss of mitochondrial membrane potential, plasma membrane rupture, and LDH release. Conversely, positive allosteric modulation of P2X7 primarily promotes an intrinsic apoptosis pathway. This was characterised by an increase in mitochondrial Ca2+, accelerated production of mitochondrial ROS, loss of mitochondrial membrane permeability in a Bax-dependent manner, the potential involvement of caspase-1, and caspase-3, and significantly accelerated kinetics of caspase-3 activation. This study highlights the ability of positive allosteric modulators to calibrate P2X7-dependent cell death pathways and may have important implications in modulating the antimicrobial immune response and in the resolution of inflammation.

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Tumor-induced apoptosis of T lymphocytes: elucidation of intracellular apoptotic events

Blood ◽

10.1182/blood.v95.6.2015 ◽

2000 ◽

Vol 95 (6) ◽

pp. 2015-2023 ◽

Cited By ~ 60

Author(s):

Brian R. Gastman ◽

Daniel E. Johnson ◽

Theresa L. Whiteside ◽

Hannah Rabinowich

Keyword(s):

T Cells ◽

Cell Death ◽

T Cell ◽

T Lymphocytes ◽

Tumor Cells ◽

Caspase 3 ◽

Jurkat Cells ◽

Caspase 8 ◽

Induced Apoptosis ◽

Fluoromethyl Ketone

Abstract Our recent studies suggest that human squamous cell carcinoma of the head and neck (SCCHN) is capable of activating an intrinsic mechanism of programmed-cell death in interacting lymphocytes in situ and in vitro. The current study used Jurkat T-cell line as a model to investigate intracellular apoptotic events in T cells interacting with SCCHN. Apoptosis induced in T lymphocytes by tumor cells was in part Fas-mediated, since it was partially, but significantly, inhibited in the presence of anti-Fas ligand Ab or in Fas-resistant Jurkat cells. The synthetic caspase inhibitors, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) and N-benzyloxycarbonyl-Asp-glu-Val-Asp-fluoromethyl ketone (Z-DEVD-FMK), effectively blocked apoptosis of Jurkat cells co-incubated with SCCHN cell lines, suggesting the involvement of caspases in tumor-induced apoptosis of lymphocytes. Overexpression of CrmA, an inhibitor of caspase-1 and caspase-8, partially inhibited tumor-induced T-cell death. Caspase-8 and caspase-3 were identified as effector molecules in the execution of tumor-induced T-cell death, since the proform enzymes were processed into active subunits during co-incubation of T cells with tumor cells. Furthermore, co-incubation with tumor cells resulted in cleavage of poly(ADP-ribose) polymerase (PARP), a common caspase-3 substrate, and in cleavage of TcR-ζ chain, shown by us to be a T-cell specific caspase-3 substrate. Overexpression of Bcl-2 did not provide protection of T cells from SCCHN-induced DNA degradation. Instead, the Bcl-2 protein was cleaved in the target T cells during their co-incubation with tumor cells. These findings demonstrate that tumor cells can trigger in T lymphocytes caspase-dependent apoptotic cascades, which are not effectively protected by Bcl-2.

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The P-MAPA immunomodulator partially prevents apoptosis induced by Zika virus infection in THP-1 cells

Current Pharmaceutical Biotechnology ◽

10.2174/1389201021666200602140005 ◽

2020 ◽

Vol 21 ◽

Cited By ~ 1

Author(s):

Morganna C. Lima ◽

Elisa A. N. Azevedo ◽

Clarice N. L. de Morais ◽

Larissa I. O. de Sousa ◽

Bruno M. Carvalho ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Death ◽

Virus Infection ◽

Virus Replication ◽

Zika Virus ◽

Mammalian Cells ◽

Caspase 3 ◽

Neurological Complications ◽

Zika Virus Infection

Background: Zika virus is an emerging arbovirus of global importance. ZIKV infection is associated with a range of neurological complications such as the Congenital Zika Syndrome and Guillain Barré Syndrome. Despite the magnitude of recent outbreaks, there is no specific therapy to prevent or to alleviate disease pathology. Objective: To investigate the role of P-MAPA immunomodulator in Zika-infected THP-1 cells. Methods: THP-1 cells were subjected at Zika virus infection (Multiplicity of Infection = 0.5) followed by treatment with P-MAPA for until 96 hours post-infection. After that, the cell death was analyzed by annexin+/ PI+ and caspase 3/ 7+ staining by flow cytometry. In addition, the virus replication and cell proliferation were accessed by RT-qPCR and Ki67 staining, respectively. Results: We demonstrate that P-MAPA in vitro treatment significantly reduces Zika virus-induced cell death and caspase-3/7 activation on THP-1 infected cells, albeit it has no role in virus replication and cell proliferation. Conclusions: Our study reveals that P-MAPA seems to be a satisfactory alternative to inhibits the effects of Zika virus infection in mammalian cells.

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Caspase-8 and Caspase-3 Are Expressed by Different Populations of Cortical Neurons Undergoing Delayed Cell Death after Focal Stroke in the Rat

Journal of Neuroscience ◽

10.1523/jneurosci.19-14-05932.1999 ◽

1999 ◽

Vol 19 (14) ◽

pp. 5932-5941 ◽

Cited By ~ 178

Author(s):

James J. Velier ◽

Julie A. Ellison ◽

Kristine K. Kikly ◽

Patricia A. Spera ◽

Frank C. Barone ◽

...

Keyword(s):

Cell Death ◽

Cortical Neurons ◽

Caspase 3 ◽

Caspase 8 ◽

Delayed Cell Death ◽

Different Populations

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Using More Than 1 (Path)Way to Kill a Host Cell: Lessons From Clostridium perfringens Enterotoxin

Microbiology Insights ◽

10.1177/1178636120931518 ◽

2020 ◽

Vol 13 ◽

pp. 117863612093151

Author(s):

Bruce McClane ◽

Archana Shrestha

Keyword(s):

Recent Work ◽

Clostridium Perfringens ◽

Caspase 3 ◽

Kinase Domain ◽

Intermediate Step ◽

Clostridium Perfringens Enterotoxin ◽

Mixed Lineage Kinase ◽

Intestinal Infections ◽

Apoptosis And Necrosis

Clostridium perfringens enterotoxin (CPE) is responsible for the symptoms of common intestinal infections due to C. perfringens type F isolates. CPE is a pore-forming toxin that uses certain claudins as a receptor. Previous studies showed that, in enterocyte-like Caco-2 cells, low CPE concentrations cause caspase 3-mediated apoptosis but high CPE concentrations cause necrosis. The recent work published in mBio by Shrestha, Mehdizadeh Gohari, and McClane determined that RIP1 and RIP3 are involved in both CPE-mediated apoptosis and necrosis in Caco-2 cells. Furthermore, mixed lineage kinase-domain (MLKL) oligomerization was shown to be important for necrosis caused by CPE, identifying this necrosis as programmed necroptosis. In addition, calpain activation due to Ca2+ influx through the CPE pore was identified as a critical intermediate step for MLKL oligomerization and, thus, CPE-induced necroptosis. These findings may have applicability to understand the action of some other pore-forming toxins that induce necroptosis and may also be important for understanding CPE action in vivo.

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The pseudokinase MLKL activates PAD4-dependent NET formation in necroptotic neutrophils

Science Signaling ◽

10.1126/scisignal.aao1716 ◽

2018 ◽

Vol 11 (546) ◽

pp. eaao1716 ◽

Cited By ~ 13

Author(s):

Akshay A. D’Cruz ◽

Mary Speir ◽

Meghan Bliss-Moreau ◽

Sylvia Dietrich ◽

Shu Wang ◽

...

Keyword(s):

Cell Death ◽

Kinase Domain ◽

Neutrophil Extracellular Trap ◽

Human Neutrophils ◽

Caspase 8 ◽

Interacting Protein ◽

Dependent Activity ◽

Mrsa Infection ◽

Death Effector ◽

Net Release

Neutrophil extracellular trap (NET) formation can generate short-term, functional anucleate cytoplasts and trigger loss of cell viability. We demonstrated that the necroptotic cell death effector mixed lineage kinase domain–like (MLKL) translocated from the cytoplasm to the plasma membrane and stimulated downstream NADPH oxidase–independent ROS production, loss of cytoplasmic granules, breakdown of the nuclear membrane, chromatin decondensation, histone hypercitrullination, and extrusion of bacteriostatic NETs. This process was coordinated by receptor-interacting protein kinase-1 (RIPK1), which activated the caspase-8–dependent apoptotic or RIPK3/MLKL-dependent necroptotic death of mouse and human neutrophils. Genetic deficiency of RIPK3 and MLKL prevented NET formation but did not prevent cell death, which was because of residual caspase-8–dependent activity. Peptidylarginine deiminase 4 (PAD4) was activated downstream of RIPK1/RIPK3/MLKL and was required for maximal histone hypercitrullination and NET extrusion. This work defines a distinct signaling network that activates PAD4-dependent NET release for the control of methicillin-resistant Staphylococcus aureus (MRSA) infection.

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Interaction with Sug1 enables Ipaf ubiquitination leading to caspase 8 activation and cell death

Biochemical Journal ◽

10.1042/bj20091349 ◽

2010 ◽

Vol 427 (1) ◽

pp. 91-104 ◽

Cited By ~ 26

Author(s):

Yatender Kumar ◽

Vegesna Radha ◽

Ghanshyam Swarup

Keyword(s):

Cell Death ◽

Mammalian Cells ◽

Intramolecular Interaction ◽

Dominant Negative ◽

26S Proteasome ◽

Caspase 8 ◽

Interacting Protein ◽

Caspase 1 ◽

Lung Carcinoma Cells ◽

Lrr Domain

Activation of initiator caspases is dependent on interacting proteins, and Ipaf [ICE (interleukin-1β-converting enzyme)-protease activating factor] {NLRC4 [NLR (Nod-like receptor) family CARD (caspase activation and recruitment domain)-containing 4]} an inflammasome component, is involved in caspase 1 activation and apoptosis. Investigating the mechanisms of Ipaf activation, we found that the C-terminal LRR (leucine-rich repeat) domain of Ipaf, through intramolecular interaction, negatively regulates its apoptosis-inducing function. In A549 lung carcinoma cells, expression of Ac-Ipaf (LRR-domain-deleted Ipaf) induced cell death that was dependent on caspase 8, but not on caspase 1. A yeast two-hybrid screen using Ac-Ipaf as bait identified human Sug1 (suppressor of gal 1), a component of the 26S proteasome, as an interacting protein. In mammalian cells Sug1 interacts and co-localizes with Ipaf. Sug1 binds to amino acids 91–253 of Ipaf, which is also the region that the LRR domain binds to. It potentiates cell death induced by Ipaf and Ac-Ipaf, and co-expression of Sug1 and Ipaf induces caspase-8-dependent cell death. Cellular complexes formed by Ipaf and Sug1 contain caspase 8. Expression of Ac-Ipaf or co-expression of Sug1 with Ipaf results in the formation of cytoplasmic aggregates and caspase 8 activation. Sug1 co-expression enabled modification of Ipaf by ubiquitination. Tagging ubiquitin molecules to Ipaf led to aggregate formation, enhanced caspase 8 interaction and activation, resulting in induction of cell death. Using RNAi (RNA interference) and dominant-negative approaches, we have shown that cell death induced by Ac-Ipaf expression or by treatment with TNF-α (tumour necrosis factor α) or doxorubicin is dependent on Sug1. Our results suggest a role for ubiquitination of Ipaf that is enabled by its interaction with Sug1, leading to caspase 8 activation and cell death.

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The Multifaceted Roles of Pyroptotic Cell Death Pathways in Cancer

Cancers ◽

10.3390/cancers11091313 ◽

2019 ◽

Vol 11 (9) ◽

pp. 1313

Author(s):

Man Wang ◽

Shuai Jiang ◽

Yinfeng Zhang ◽

Peifeng Li ◽

Kun Wang

Keyword(s):

Cell Death ◽

Biological Significance ◽

Chemotherapeutic Agents ◽

The Body ◽

Cell Swelling ◽

Pathogen Invasion ◽

Membrane Rupture ◽

Cancer Management ◽

Cell Death Pathways ◽

Cancer Pathogenesis

Cancer is a category of diseases involving abnormal cell growth with the potential to invade other parts of the body. Chemotherapy is the most widely used first-line treatment for multiple forms of cancer. Chemotherapeutic agents act via targeting the cellular apoptotic pathway. However, cancer cells usually acquire chemoresistance, leading to poor outcomes in cancer patients. For that reason, it is imperative to discover other cell death pathways for improved cancer intervention. Pyroptosis is a new form of programmed cell death that commonly occurs upon pathogen invasion. Pyroptosis is marked by cell swelling and plasma membrane rupture, which results in the release of cytosolic contents into the extracellular space. Currently, pyroptosis is proposed to be an alternative mode of cell death in cancer treatment. Accumulating evidence shows that the key components of pyroptotic cell death pathways, including inflammasomes, gasdermins and pro-inflammatory cytokines, are involved in the initiation and progression of cancer. Interfering with pyroptotic cell death pathways may represent a promising therapeutic option for cancer management. In this review, we describe the current knowledge regarding the biological significance of pyroptotic cell death pathways in cancer pathogenesis and also discuss their potential therapeutic utility.

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Analysis of the N-terminal region of human MLKL, as well as two distinct MLKL isoforms, reveals new insights into necroptotic cell death

Bioscience Reports ◽

10.1042/bsr20150246 ◽

2016 ◽

Vol 36 (1) ◽

Cited By ~ 9

Author(s):

Katja Hrovat Arnež ◽

Michaela Kindlova ◽

Nilesh J. Bokil ◽

James M. Murphy ◽

Matthew J. Sweet ◽

...

Keyword(s):

Cell Death ◽

Human Monocyte ◽

Kinase Domain ◽

Terminal Region ◽

Activation Loop ◽

Phosphorylation Sites ◽

Mixed Lineage Kinase

We show that mixed lineage kinase domain-like (MLKL) isoform 2, which lacks the pseudokinase domain and activation loop phosphorylation sites, is a more potent activator of cell death compared with MLKL isoform 1. Both MLKL isoforms are expressed in human monocyte-derived macrophages.

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SMAC-Mimetic BV-6 Sensitizes Therapeutic Agents-Induced Apoptosis In AML Cells

Blood ◽

10.1182/blood.v116.21.2177.2177 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2177-2177

Author(s):

Duncan H Mak ◽

Christa Manton ◽

Michael Andreeff ◽

Bing Z Carter

Keyword(s):

Cell Death ◽

Vector Control ◽

Caspase 3 ◽

Jurkat Cells ◽

Leukemic Cells ◽

Caspase 8 ◽

Caspase 9 ◽

Smac Mimetic ◽

P53 Activation ◽

Induced Apoptosis

Abstract Abstract 2177 The antiapoptotic function of the inhibitors of apoptosis family of proteins (IAPs) is antagonized by mitochondria-released SMAC protein. The IAP-member XIAP suppresses apoptosis by directly binding and inhibiting caspase-9 and caspase-3, while cIAP1, a component of the cytoplasmic signaling complex containing TNF receptor associated factors, suppresses apoptosis via the caspase-8-mediated pathway. BV-6 (Genentech) is a bivalent SMAC-mimetic and has been shown to promote cell death by inducing cIAP autoubiquitination, NF-κB activation, and TNFα-dependent apoptosis. We examined its effect on leukemic cells and found that BV-6 only moderately induced apoptosis. The EC50 was found to be 15.3±5.1 μM at 48 hours in OCI-AML3 cells which are relatively sensitive. We then determined whether BV-6 sensitizes leukemic cells to the HDM2-inhibitor nutlin-3a and to Ara-C. p53 modulates the expression and activity of Bcl-2 family proteins and promotes the mitochondrial-mediated apoptosis. We showed previously that activation of p53 by nutlin-3a sensitizes AML cells to XIAP inhibition induced-death in part by promoting the release of SMAC from mitochondrion (Carter BZ et al., Blood 2010). We treated OCI-AML3 cells with BV-6, nutlin-3a or Ara-C, and BV-6+nutlin-3a or BV-6+Ara-C and found that the combination of BV-6 and nutlin-3a or BV-6 and Ara-C synergistically induced cell death in OCI-AML3 cells with a combination index (CI) of 0.27±0.11 and 0.22±0.05 (48 hours), respectively. To demonstrate that p53 activation is essential for the synergism of BV-6+nutlin-3a combination, we treated OCI-AML3 vector control and p53 knockdown cells with these two agents and found that the combination synergistically promoted cell death in the vector control (CI=0.47±0.15) but not in the p53 knockdown cells, as expected, while BV6+Ara-C was synergistic in both vector control and p53 knockdown cells (CI=0.15±0.03 and 0.08±0.03, respectively, 48 hours). BV-6 induced activation of caspase-8, caspase-9, and caspase-3 and decreased XIAP levels, but did not cause rapid cIAP1 degradation, as reported by others. To assess the contribution of death receptor-mediated apoptosis in BV-6-induced cell death, we treated Jurkat and caspase-8 mutated Jurkat cells (JurkatI9.2) with BV-6 and found that BV-6 induced cell death and significantly potentiated TRAIL-induced apoptosis in Jurkat cells (CI=0.14±0.08, 48 hours). Caspase-8 mutated JurkatI9.2 cells were significantly less sensitive to BV-6 than Jurkat cells and as expected, JurkatI9.2 was completely resistant to TRAIL. Collectively, we showed that the bivalent SMAC-mimetic BV-6 potentiates p53 activation-, chemotherapy-, and TRAIL-induced cell death, but has only minimal activity by itself in leukemic cells. SMAC-mimetics could be useful in enhancing the efficacy of different classes of therapeutic agents used in AML therapy. Disclosures: No relevant conflicts of interest to declare.

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