Caspase Inhibitor z-DEVD-fmk Attenuates Calpain and Necrotic Cell Death in Vitro and after Traumatic Brain Injury

In studies designed to evaluate the therapeutic window for treatment of traumatic brain injury, the caspase 3 inhibitor z-DEVD-fmk improved neurologic function and reduced lesion volumes when administered at 1 but not at 4, 8, or 24 hours after injury. Moreover, neither caspase 3 nor PARP, a caspase 3 substrate, were cleaved in injured, untreated cortex from 1 to 72 hours after injury. Few cortical neurons expressed active caspase 3 or were TUNEL positive from 6 to 24 hours after injury, and TUNEL staining was primarily Type I (necrotic). Nissl staining revealed extensive neuronal necrosis in the injured cortex from 6 to 24 hours after impact. Considered together, these data suggested that z-DEVD-fmk may reduce neuronal necrosis, so we used an in vitro model of necrotic cell death induced by maitotoxin to test this further and explore the potential mechanism(s) involved. Z-DEVD-fmk (1 nM-100 μM) significantly attenuated maitotoxin induced neuronal cell death and markedly reduced expression of the 145 kD calpain-mediated α-spectrin breakdown product after maitotoxin injury. Neither the 120 kD caspase-mediated α-spectrin cleavage product nor cathepsin B were expressed after maitotoxin injury. In a cell free assay, z-DEVD-fmk reduced hydrolysis of casein by purified calpain I. Finally, z-DEVD-fmk reduced expression of the 145 kD calpain-mediated α-spectrin cleavage fragment after traumatic brain injury in vivo. These data suggest that neuroprotection by z-DEVD-fmk may, in part, reflect inhibition of calpain-related necrotic cell death.

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Targeting Necrosis: Elastase-like Protease Inhibitors Curtail Necrotic Cell Death Both In Vitro and in Three In Vivo Disease Models

Journal of Medicinal Chemistry ◽

10.1021/acs.jmedchem.0c01683 ◽

2021 ◽

Vol 64 (3) ◽

pp. 1510-1523

Author(s):

Boris Khalfin ◽

Alexandra Lichtenstein ◽

Amnon Albeck ◽

Ilana Nathan

Keyword(s):

Cell Death ◽

Protease Inhibitors ◽

Disease Models ◽

Necrotic Cell Death ◽

Necrotic Cell

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Repurposing Cationic Amphiphilic Drugs and Derivatives to Engage Lysosomal Cell Death in Cancer Treatment

Frontiers in Oncology ◽

10.3389/fonc.2020.605361 ◽

2020 ◽

Vol 10 ◽

Author(s):

Michelle Hu ◽

Kermit L. Carraway

Keyword(s):

Cell Death ◽

Tumor Cell ◽

Cellular Transformation ◽

Therapeutic Window ◽

Cell Populations ◽

Necrotic Cell Death ◽

Necrotic Cell ◽

Membrane Rupture ◽

Cationic Amphiphilic Drugs ◽

Amphiphilic Drugs

A major confounding issue in the successful treatment of cancer is the existence of tumor cell populations that resist therapeutic agents and regimens. While tremendous effort has gone into understanding the biochemical mechanisms underlying resistance to each traditional and targeted therapeutic, a broader approach to the problem may emerge from the recognition that existing anti-cancer agents elicit their cytotoxic effects almost exclusively through apoptosis. Considering the myriad mechanisms cancer cells employ to subvert apoptotic death, an attractive alternative approach would leverage programmed necrotic mechanisms to side-step therapeutic resistance to apoptosis-inducing agents. Lysosomal cell death (LCD) is a programmed necrotic cell death mechanism that is engaged upon the compromise of the limiting membrane of the lysosome, a process called lysosomal membrane permeabilization (LMP). The release of lysosomal components into the cytosol upon LMP triggers biochemical cascades that lead to plasma membrane rupture and necrotic cell death. Interestingly, the process of cellular transformation appears to render the limiting lysosomal membranes of tumor cells more fragile than non-transformed cells, offering a potential therapeutic window for drug development. Here we outline the concepts of LMP and LCD, and discuss strategies for the development of agents to engage these processes. Importantly, the potential exists for existing cationic amphiphilic drugs such as antidepressants, antibiotics, antiarrhythmics, and diuretics to be repurposed to engage LCD within therapy-resistant tumor cell populations.

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Determination of apoptotic and necrotic cell death in vitro and in vivo

Methods ◽

10.1016/j.ymeth.2013.02.011 ◽

2013 ◽

Vol 61 (2) ◽

pp. 117-129 ◽

Cited By ~ 130

Author(s):

Tom Vanden Berghe ◽

Sasker Grootjans ◽

Vera Goossens ◽

Yves Dondelinger ◽

Dmitri V. Krysko ◽

...

Keyword(s):

Cell Death ◽

Necrotic Cell Death ◽

Necrotic Cell

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17Beta-estradiol attenuates programmed cell death in cortical pericontusional zone following traumatic brain injury via upregulation of ERalpha and inhibition of caspase-3 activation

Neurochemistry International ◽

10.1016/j.neuint.2010.11.006 ◽

2011 ◽

Vol 58 (1) ◽

pp. 126-133 ◽

Cited By ~ 13

Author(s):

Li-Zhuo Li ◽

Yi-Jun Bao ◽

Min Zhao

Keyword(s):

Traumatic Brain Injury ◽

Cell Death ◽

Brain Injury ◽

Programmed Cell Death ◽

Caspase 3

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CASPASE-3 MEDIATED PROGRAMMED-CELL DEATH (APOPTOSIS) AFTER TRAUMATIC BRAIN INJURY IN RATS

Critical Care Medicine ◽

10.1097/00003246-199901001-00095 ◽

1999 ◽

Vol 27 (Supplement) ◽

pp. 53A

Author(s):

Robert S. B. Clark ◽

Patrick M. Kochanek ◽

Simon C. Watkins ◽

Minzhi Chen ◽

Neal A. Seidberg ◽

...

Keyword(s):

Traumatic Brain Injury ◽

Cell Death ◽

Brain Injury ◽

Programmed Cell Death ◽

Caspase 3

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Concurrent Assessment of Calpain and Caspase-3 Activation after Oxygen–Glucose Deprivation in Primary Septo-Hippocampal Cultures

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/00004647-200111000-00004 ◽

2001 ◽

Vol 21 (11) ◽

pp. 1281-1294 ◽

Cited By ~ 47

Author(s):

Jennifer K. Newcomb-Fernandez ◽

Xiurong Zhao ◽

Brian R. Pike ◽

Kevin K. W. Wang ◽

Andreas Kampfl ◽

...

Keyword(s):

Cell Death ◽

Caspase 3 ◽

Apoptotic Cell ◽

Glucose Deprivation ◽

Apoptotic Cell Death ◽

Oxygen Glucose Deprivation ◽

Necrotic Cell Death ◽

Necrotic Cell ◽

Lactate Dehydrogenase Release ◽

Hippocampal Cultures

The contributions of calpain and caspase-3 to apoptosis and necrosis after central nervous system (CNS) trauma are relatively unexplored. No study has examined concurrent activation of calpain and caspase-3 in necrotic or apoptotic cell death after any CNS insult. Experiments used a model of oxygen–glucose deprivation (OGD) in primary septo-hippocampal cultures and assessed cell viability, occurrence of apoptotic and necrotic cell death phenotypes, and protease activation. Immunoblots using an antibody detecting calpain and caspase-3 proteolysis of α-spectrin showed greater accumulation of calpain-mediated breakdown products (BDPs) compared with caspase-3–mediated BDPs. Administration of calpain and caspase-3 inhibitors confirmed that activation of these proteases contributed to cell death, as inferred by lactate dehydrogenase release. Oxygen–glucose deprivation resulted in expression of apoptotic and necrotic cell death phenotypes, especially in neurons. Immunocytochemical studies of calpain and caspase-3 activation in apoptotic cells indicated that these proteases are almost always concurrently activated during apoptosis. These data demonstrate that calpain and caspase-3 activation is associated with expression of apoptotic cell death phenotypes after OGD, and that calpain activation, in combination with caspase-3 activation, could contribute to the expression of apoptotic cell death by assisting in the degradation of important cellular proteins.

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Inhibition of ?-ketoglutarate dehydrogenase complex promotes cytochromec release from mitochondria, caspase-3 activation, and necrotic cell death

Journal of Neuroscience Research ◽

10.1002/jnr.10756 ◽

2003 ◽

Vol 74 (2) ◽

pp. 309-317 ◽

Cited By ~ 50

Author(s):

Hsueh-Meei Huang ◽

Hsiu-Chong Ou ◽

Hui Xu ◽

Huan-Lian Chen ◽

Corinne Fowler ◽

...

Keyword(s):

Cell Death ◽

Caspase 3 ◽

Necrotic Cell Death ◽

Necrotic Cell ◽

Dehydrogenase Complex ◽

Ketoglutarate Dehydrogenase

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Abstract 23: Disruption of TRAF2-TAK1-NF-κB Signaling Axis Triggers K-48 Linked Poly-Ubiquitylation of RIP1 and Necrotic Cell Death in Doxorubicin Cardiotoxicity

Circulation Research ◽

10.1161/res.117.suppl_1.23 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Rimpy Dhingra ◽

Victoria Margulets ◽

Floribeth Aguilar ◽

Lorrie A. Kirshenbaum

Keyword(s):

Cell Death ◽

Cardiac Myocytes ◽

Cell Injury ◽

Ventricular Myocytes ◽

Necrotic Cell Death ◽

Ros Production ◽

Necrotic Cell ◽

Interacting Protein

The anthracycline doxorubicin (Dox) is a highly effective anti-tumour agent, however, its use is limited by its severe cardiotoxic effects that manifests as heart failure. The decline in cardiac performance induced by doxorubicin remains poorly defined. A critical survival role for the canonical IKKβ -mTOR-NF-κB signaling pathway has been demonstrated in ventricular myocytes. In this report, we demonstrate that, Dox impairs IKKβ-mTOR- NF-κB signaling in ventricular myocytes accompanied by mitochondrial perturbations including mPTP, loss of mitochondrial membrane potential and ROS production. IKKβ- NF-κB signaling involves TRAF 2 mediated ligation of K63- ubiquitin chains to RIP1 (Receptor Interacting Protein 1) which serves as scaffold for recruitment of ubiquitylated Tak1 complexes and phosphorylation-dependent activation of IKKβ -NF-kB signaling. Interestingly, ventricular myocytes treated with dox demonstrated reduction in expression levels of TRAF2 and TAK1, in vivo and in vitro. This was accompanied by a decline in K63- and concomitant increase in K-48 linked polyubiquitination on RIP1, impaired NF-kB activation and necrotic cell death of cardiac myocytes. Interestingly, inhibiting the kinase activity of RIP1 with Necrostatin-1, (Nec1) suppressed necrotic cell injury induced by dox but not NF-kB activation. Concordant with these findings was a marked increase in necrotic cell death in cardiac myocytes defective for IKKB signaling or MEF cells deficient for p65 treated with dox. Notably, mitochondrial perturbations, including PT-pore opening , ROS production, calcium uptake, LDH, Tn(T) and HMGB-1 release and necrotic cell injury induced by dox were completely abrogated by restoring NF-kB signaling in cardiac myocytes or Nec-1. Herein, we provide novel evidence that K-48 linked poly ubiquitylation of RIP1 provides a functional switch that impairs NF-kB activation and signals necrosis in cells treated with dox. Interventions that modulate NF-kB activity may prove beneficial in mitigating the cardiotoxic effects of dox.

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