A secreted caspase-3-substrate-cleaving activity at low pH belongs to cathepsin B: a study on primary brain cell cultures

Онуфриев, М. В.; Yakovlev, A. A.; Lyzhin, A. A.; Stepanichev, M. Yu.; Хаспеков, Л. Г.; Gulyaeva, N. V.

doi:10.1134/s0006297909030067

Cited by 11 publications

(4 citation statements)

References 32 publications

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“…Fluorophore-labelled caspase-cleavable probes (for example, DEVD) are also commonly utilized despite reports of differential or attenuated cleavage when compared to physiological caspase substrates as well as activation by non-caspase proteases. 7, 8 Moreover, many laboratories employ additional secondary processing steps (for example, flow cytometry methods to count cells in each well) following the acquisition of high-content live-cell imaging data due to a lack of validated protocols controlling for inter-well plating variability and proliferation changes due to treatments. Collectively, these practices undermine the high-throughput nature of live-cell imagers and are limited by the commercially available reporters.…”

mentioning

confidence: 99%

Robust high-throughput kinetic analysis of apoptosis with real-time high-content live-cell imaging

Gelles

Chipuk

2016

Cell Death Dis

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Quantitative and kinetic analyses of apoptotic cell death are integral components of exploring cell biology, measuring cellular stress responses, and performing high-throughput genomic/RNAi/drug screens. Here, we present a detailed method that integrates robust kinetic real-time high-content imaging with Annexin V labelling to provide a highly sensitive, accurate, simple and zero-handling approach to quantify extrinsic and intrinsic inducers of apoptosis. The sensitivity of this non-toxic method outperforms previous high-throughput methodologies using viability dyes or caspase-activation reporters. This method also incorporates a multiplex adaptation to integrate variability in cell number due to treatment-induced proliferation changes and the detachment of dying cells. Compared to Annexin V detection by flow cytometry, this method is 10-fold more sensitive, eliminates extensive sample handling and processing, and provides real-time kinetics of apoptosis at both single-cell and population-level resolutions.

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mentioning

confidence: 99%

Robust high-throughput kinetic analysis of apoptosis with real-time high-content live-cell imaging

Gelles

Chipuk

2016

Cell Death Dis

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“…Under pathological conditions, such as hypoperfusion or ischemia, activation of cell death-related proteases may result in destruction of the cytoskeleton during the early stages of cell demise, which promotes detachment of neurons from their surrounding cells (Böhm, 2003). Furthermore, these enzymes may be secreted by neurons (Onufriev et al, 2009) and cleave their substrates in the extracellular space, thus facilitating migration of cells to the stratum lucidum.…”

Section: Discussionmentioning

confidence: 99%

Cholinergic Deficit Induced by Central Administration of 192IgG-Saporin Is Associated With Activation of Microglia and Cell Loss in the Dorsal Hippocampus of Rats

et al. 2019

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Alzheimer’s disease (AD) is associated with degeneration of cholinergic neurons in the basal forebrain. Administration of the immunotoxin 192IgG-saporin to rats, an animal model of AD, leads to degeneration of cholinergic neurons in the medial septal area. In the present study, cholinergic cell death was induced by intracerebroventricular administration of 192IgG-saporin. One and a half months after injection, we studied the histopathology of the hippocampus and the responses of microglia and astrocytes using immunohistochemistry and neuroglial gene expression. We found that treatment with 192IgG-saporin resulted in neuronal loss in the CA3 field of the hippocampus. Microglial proliferation was observed in the dentate gyrus of the dorsal hippocampus and white matter. Massive proliferation and activation of microglia in the white matter was associated with strong activation of astrocytes. However, the expression of microglial marker genes significantly increased only in the dorsal hippocampus, not the ventral hippocampus. These effects were not related to non-specific action of 192IgG-saporin because of the absence of the Nerve growth factor receptor in the hippocampus. Additionally, 192IgG-saporin treatment also induced a decrease in the expression of genes that are associated with transport functions of brain vascular cells (Slc22a8, Ptprb, Sdpr), again in the dorsal hippocampus but not in the ventral hippocampus. Taken together, our data suggest that cholinergic degeneration in the medial septal area induced by intracerebroventricular administration of 192IgG-saporin results in an increase in the number of microglial cells and neuron degeneration in the dorsal hippocampus.

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“…In fact, it has been suggested that the clinical failures of the attempts to selectively deliver drugs to the tumor that has been successful in preclinical models were due to the heterogeneous tumor in human patients while the tumors established in the preclinical models are largely homogeneous (ref). Moreover, Interestingly, DEVD has been reported to be hydrolyzed by cathepsin B at pH 5–6, suggesting that prodrug systems such as ADC that are internalized into the cells by endocytosis could easily take advantage of the STAEPT paradigm by simply replacing the linker with DEVD. In conclusion, we believe that STAEPT could be a promising strategy to improve upon current targeted therapies, especially the ones that exploit cytotoxins as their warhead.…”

Section: Resultsmentioning

confidence: 99%

Self‐Triggered Apoptosis Enzyme Prodrug Therapy (STAEPT): Enhancing Targeted Therapies via Recurrent Bystander Killing Effect by Exploiting Caspase‐Cleavable Linker

et al. 2018

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Tumor heterogeneity is associated with the therapeutic failures of targeted therapies. To overcome such heterogeneity, a novel targeted therapy is proposed that could kill tumor populations with diverse phenotypes by delivering nonselective cytotoxins to target‐positive cells as well as to the surrounding tumor cells via a recurrent bystander killing effect. A representative prodrug is prepared that targets integrin αvβ3 and releases cytotoxins upon entering cells or by caspase‐3. This allows the prodrug to kill integrin αvβ3‐positive cells and upregulate caspase‐3, which in turn, activates the prodrug to release a cytotoxin that could subsequently diffuse into and kill the neighboring tumor cells. Apoptotic cells further upregulate and release caspase‐3, which activate more prodrugs leading to another round of adjacent cell death and caspase‐3 release. Thus, the bystander killing effect could occur repeatedly, leading to augmented and widespread anticancer activity. This strategy provides an avenue that could advance the current targeted therapy.

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A secreted caspase-3-substrate-cleaving activity at low pH belongs to cathepsin B: a study on primary brain cell cultures

Cited by 11 publications

References 32 publications

Robust high-throughput kinetic analysis of apoptosis with real-time high-content live-cell imaging

Robust high-throughput kinetic analysis of apoptosis with real-time high-content live-cell imaging

Cholinergic Deficit Induced by Central Administration of 192IgG-Saporin Is Associated With Activation of Microglia and Cell Loss in the Dorsal Hippocampus of Rats

Self‐Triggered Apoptosis Enzyme Prodrug Therapy (STAEPT): Enhancing Targeted Therapies via Recurrent Bystander Killing Effect by Exploiting Caspase‐Cleavable Linker

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