scholarly journals Certain protein transducing agents convert translocated proteins into cell killers.

2012 ◽  
Vol 59 (3) ◽  
Author(s):  
Siergiej Tcherniuk ◽  
Anne-Laure Fiser ◽  
Madiha Derouazi ◽  
Bertrand Toussaint ◽  
Yan Wang ◽  
...  
Keyword(s):  
Cell Death ◽  
Cytotoxic Effect ◽  
Cultured Cells ◽  
Large Range ◽  
Protein Transduction ◽  
Cell Interior ◽  
Cell Penetrating ◽  
Translocated Proteins ◽  
Control Protein

The majority of proteins are unable to translocate into the cell interior. Hence for peptide- and protein-based therapeutics a direct intracytoplasmic delivery with the aid of transducing agents is an attractive approach. We wanted to deliver to the cell interior a putatively cytotoxic protein VPg. Protein transduction was achieved in vitro with three different commercial products. However, in our hands, delivery of various control proteins without known deleterious effects, as well as of protein VPg, always induced cell death. Finally, we used a novel transducing peptide Wr-T, which was not toxic to cultured cells, even in a quite large range of concentrations. Most importantly, control protein delivered to cells in culture did not display any toxicity while VPg protein exerted a strong cytotoxic effect. These data show that results obtained with cell-penetrating agents should be interpreted with caution.

scholarly journals Dominant-negative ATF5 rapidly depletes survivin in tumor cells

2019 ◽  
Vol 10 (10) ◽  
Author(s):  
Xiaotian Sun ◽  
James M. Angelastro ◽  
David Merino ◽  
Qing Zhou ◽  
Markus D. Siegelin ◽  
...  
Keyword(s):  
Cell Death ◽  
Tumor Cell ◽  
Cancer Cells ◽  
Dominant Negative ◽  
Tumor Cell Death ◽  
Cell Penetrating ◽  
Selective Death ◽  
Tumor Types

Abstract Survivin (BIRC5, product of the BIRC5 gene) is highly expressed in many tumor types and has been widely identified as a potential target for cancer therapy. However, effective anti-survivin drugs remain to be developed. Here we report that both vector-delivered and cell-penetrating dominant-negative (dn) forms of the transcription factor ATF5 that promote selective death of cancer cells in vitro and in vivo cause survivin depletion in tumor cell lines of varying origins. dn-ATF5 decreases levels of both survivin mRNA and protein. The depletion of survivin protein appears to be driven at least in part by enhanced proteasomal turnover and depletion of the deubiquitinase USP9X. Survivin loss is rapid and precedes the onset of cell death triggered by dn-ATF5. Although survivin downregulation is sufficient to drive tumor cell death, survivin over-expression does not rescue cancer cells from dn-ATF5-promoted apoptosis. This indicates that dn-ATF5 kills malignant cells by multiple mechanisms that include, but are not limited to, survivin depletion. Cell-penetrating forms of dn-ATF5 are currently being developed for potential therapeutic use and the present findings suggest that they may pose an advantage over treatments that target only survivin.

scholarly journals Antioxidant and Proapoptotic Activities ofSclerocarya birrea[(A. Rich.) Hochst.] Methanolic Root Extract on the Hepatocellular Carcinoma Cell Line HepG2

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Maria Francesca Armentano ◽  
Faustino Bisaccia ◽  
Rocchina Miglionico ◽  
Daniela Russo ◽  
Nicoletta Nolfi ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
Hepg2 Cells ◽  
Cytotoxic Effect ◽  
Antioxidant Activities ◽  
Dermal Fibroblasts ◽  
Root Extract ◽  
Dependent Manner ◽  
Mitochondrial Membrane Depolarization

The main goal of this study was to characterize thein vitroantioxidant activity and the apoptotic potential ofS. birreamethanolic root extract (MRE). Among four tested extracts, obtained with different solvents, MRE showed the highest content of polyphenols, flavonoids, and tannins together with antioxidant activities tested with superoxide, nitric oxide, ABTS, and beta-carotene bleaching assays. Moreover, the cytotoxic effect of MRE was evaluated on the hepatocarcinoma cell line HepG2. In these cells, MRE treatment induced apoptosis and generated reactive oxygen species (ROS) in dose-dependent manner. The cytotoxic effect promoted by MRE was prevented by pretreatment of HepG2 cells with N-acetyl-L-cysteine (NAC), suggesting that oxidative stress was pivotal in MRE-mediated cell death. Moreover, we showed that the MRE treatment induced the mitochondrial membrane depolarization and the cytochromecrelease from mitochondria into the cytosol. It suggests that the apoptosis occurred in a mitochondrial-dependent pathway. Interestingly, MRE showed a sensibly lower cytotoxicity, associated with a low increase of ROS, in normal human dermal fibroblasts compared to HepG2 cells. It is suggested that the methanolic root extract ofS. Birreais able to selectively increase intracellular ROS levels in cancer cells, promoting cell death.

scholarly journals Exhaled breath condensates from healthy children induce cell death of in vitro cultured cells by activation of apoptosis

2019 ◽  
Author(s):  
Alicja Krejner-Bienias ◽  
Katarzyna Grzela ◽  
Wioletta Zagórska ◽  
Magdalena Chojnowska ◽  
Tomasz Grzela
Keyword(s):  
Cell Death ◽  
Cultured Cells ◽  
Exhaled Breath ◽  
Healthy Children ◽  
Induce Cell Death

scholarly journals Evaluation of acetone cyanohydrin effect in "in vitro" inativation of the Ehrlich ascites tumor cells

2010 ◽  
Vol 25 (1) ◽  
pp. 111-116 ◽  
Author(s):  
Rondon Tosta Ramalho ◽  
Ricardo Dutra Aydos ◽  
Marney Pascoli Cereda
Keyword(s):  
Cell Death ◽  
Tumor Cells ◽  
Cytotoxic Effect ◽  
Human Lymphocytes ◽  
Ehrlich Ascites Tumor ◽  
Ehrlich Ascites Tumor Cells ◽  
Ascites Tumor ◽  
Acetone Cyanohydrin ◽  
Ehrlich Ascites

PURPOSE: To evaluate the antitumor effect of acetone cyanohydrin in Ehrlich ascites tumor cells in vitro. METHODS: The Ehrlich ascites tumor cells and lymphocytes were incubated with different concentrations of acetone cyanohydrin (0, 0.5, 1.0, 2.0, 10.0, 20.0 and 30.0 μg.mL-1), After 1, 2, 3, 4, 18 and 24 hours cell viability tests were performed by the trypan blue method. RESULTS: The results demonstrated a dose-dependent cytotoxic effect against the cells of Ehrlich ascites tumor. The concentrations of 20 and 30 μg.mL-1 was 100% of cell death in only 1 and 2 hours respectively. In lower doses of 0.5, 1.0 and 2.0 μg.mL-1 the cytotoxic effect was less intense, increasing gradually with time. CONCLUSIONS: At low concentrations of 0.5, 1.0 and 2.0 μg.mL-1, more than 90% of cell death was observed only after 24 hours of incubation which is the evidence that the tumor cell has the ability to poison cumulatively and irreversibly itself with the acetone cyanohydrin when compared with the results presented by human lymphocytes that the same doses and at the same time of incubation reached a maximum of 30% of cell death, suggesting an activity of rhodanese differentiated between the two cells.

Cell Penetrating Mechanism of Bax Inhibiting Peptides.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4298-4298
Author(s):  
Jose A. Gomez ◽  
Tomoyuki Yoshida ◽  
Minh Lam ◽  
Clark W. Distelhorst ◽  
Shigemi Matsuyama
Keyword(s):  
Cell Death ◽  
Plasma Membrane ◽  
Fluorescent Dyes ◽  
Cultured Cells ◽  
Cell Penetrating Peptides ◽  
Dependent Manner ◽  
Cell Penetration ◽  
New Members ◽  
Cell Penetrating ◽  
High Degree

Abstract Plasma membrane is known to have a high degree of selectivity for molecular trafficking, and it does not allow the penetration of peptides larger than 3 amino acids. Previously known exceptions of large peptides that penetrate the plasma membrane are the Arginine rich peptides such as human immunodeficiency virus (HIV)-tat peptides. However, the mechanism of cell penetration of these peptides is largely unknown. Bax Inhibiting Peptides (BIP) are penta-peptides derived from the Bax binding domain of Ku70. At present, three types of BIP have been developed. Those are: VPMLK, VPTLK, and VPALR. All of these three BIPs directly bind Bax and inhibit Bax-mediated cell death in cultured cells as well as in animal study. Surprisingly, BIPs are cell permeable and autonomously enter the cytoplasm of the cells within 1 hr. Therefore BIPs are recognized as new members of cell penetration peptides. In this study, we investigated the mechanism of cell penetration of BIPs. DAMI cells (a human megakaryocyte cell line) and HeLa cells were used to investigate the detailed mechanism of cell penetration of BIPs. To detect the cell entry of BIPs, fluorescent dyes (fluorescein or tetramethylrodhamine) were conjugated to the N-terminus of BIPs and the cytoplasmic localization of BIPs was confirmed by confocal microscopy. Cell Penetration activities of BIPs were detected at 1 uM concentration in the culture medium. The significant accumulation of BIPs in the cytoplasm were detected within 1 hour of incubation both at 4 °C and 37 °C, suggesting that ATP-independent mechanism of cell penetration of BIP exists. However, cellular uptake of BIPs reaches plateau at 100 uM at 4 °C, whereas it increases in a dose dependent manner up to 1 mM at 37 °C without any sign of cytotoxicity. These results suggest that there are at least two mechanisms contributing to the cell penetration of BIPs that are, “ATP-independent (4 °C)” and “ATP-dependent (37 °C)” mechanisms. In addition to BIPs, we generated a series of mutated BIPs that do not bind Bax but retain cell-penetrating activities. We performed competition assay using fluorescence dye-labeled and non-labeled BIP (and the mutant BIPs), and the preliminary results suggest that there is a specific receptor for each peptide for its delivery into the cells. Our data also indicates that BIPs can deliver a cargo molecule (e.g. fluorescent dye) with at least the same molecular weight. Unlike other cell penetrating peptides, BIP has minimum toxicity due to its nature to inhibit Bax-mediated cell death. Along with the new data showing that BIP protects cells from pathological damages in cell culture and animal model, we will discuss the potential application of BIPs as a new type of drug delivery tool.

Platelet Death.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. sci-40-sci-40
Author(s):  
Emma C. Josefsson ◽  
Simone Schoenwaelder ◽  
Michael White ◽  
Matthew Goschnick ◽  
Andrew W. Roberts ◽  
...  
Keyword(s):  
Cell Death ◽  
Cultured Cells ◽  
Preliminary Evidence ◽  
Human Platelets ◽  
Mitochondrial Damage ◽  
Intrinsic Apoptosis ◽  
Apoptosis Pathway ◽  
Intrinsic Apoptosis Pathway

Abstract Human platelets exhibit a circulating lifespan of ~10 days, mouse platelets ~5 days. This finite existence is circumscribed by members of the Bcl-2 family of proteins, which control the intrinsic apoptosis pathway. Pro-survival Bcl-xL is the critical regulator of platelet lifespan, functioning to keep pro-death Bak and Bax in check, thereby maintaining platelet viability. After 5–10 days in the circulation, platelets not consumed in hemostatic processes initiate a Bak and Bax-dependent cell death program and clearance from the bloodstream. Mutations in Bcl-xL reduce platelet lifespan in a dose-dependent fashion, while deletion of Bak and Bax extend it. Studies with the BH3 mimetic compound ABT-737, which inhibits pro-survival Bcl-xL, have shown that platelets induced to undergo cell death in vitro exhibit many of the hallmarks of apoptosis in nucleated cells, including mitochondrial damage, caspase activation and externalization of membrane phosphatidylserine (PS). Whether any of these features occur during physiological platelet clearance remains unclear. Certainly, mitochondrial damage can reduce the recovery of transfused platelets, but whether PS – which is known to promote the pro-coagulant activity of agonist-activated platelets – also acts as a clearance signal for dying platelets in vivo is yet to be established. Conversely, Bak and Bax may play a role in mediating PS exposure triggered by activation. Supporting the idea that there may be crosstalk between classical platelet signaling pathways and the intrinsic apoptosis pathway is recent evidence that platelet agonists can also activate caspases. Intriguingly, elements of the intrinsic pathway may also contribute to the generation of platelets by megakaryocytes. Several groups have demonstrated that megakaryocytes contain activated caspases and that their inhibition can block platelet shedding by cultured cells. Preliminary evidence we have generated suggests that Bcl-2 family proteins may be required for platelet production in vivo. Thus, it appears that there is much to be understood about the role of the intrinsic apoptosis pathway in the regulation of platelet biogenesis, function, and death.

Al amyloidosis with cardiac involvement: cardiotoxicity of aggregation prone peptides deriving from variable domains of immunoglobulin light chains

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
P.E Nikolaou ◽  
G.I Nasi ◽  
I Sulaiman ◽  
P Spatharas ◽  
S Kikionis ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Death ◽  
Light Chain ◽  
Hot Spots ◽  
Cytotoxic Effect ◽  
Light Chains ◽  
Al Amyloidosis ◽  
H9c2 Cells ◽  
Variable Domains

Abstract Background/Introduction Light chain (AL) amyloidosis is an uncommon malignancy manifested by systemic extracellular deposition of immunoglobulin light chain fibrils. The cardiac phenotype is characterised by ventricular wall thickening and stands as the most prominent cause of morbidity and mortality. Although, it has been established that the circulating light chains directly impair cardiomyocyte function, the cytotoxic effect of specific amyloidogenic peptides that may appear due to excessive cleavage of light chains remains unspecified. Purpose In the present work, we aimed to detect amyloidogenic “hot-spots” on the variable domains of light chains associated with cardiac AL amyloidosis (IGLV1-44 and IGLV3-01) or inferior outcomes (IGLV6-57) and define their cytotoxic effect in vitro. Methods At first, we used the curated database ALBase and we performed a multiple sequence alignment of the IGLV1-44, IGLV3-01 and IGLV6-57 inputs that derived only from patients with AL amyloidosis. “Aggregation-prone” hot-spots in the conserved amino acid sequences were identified with the aid of AMYLPRED2, a tool which combines 11 independent computational methods and provides a consensus result of potent amyloidogenic regions. Five peptides were rationally selected and synthetically produced in order to be tested in vitro. The amyloidogenic properties of the peptides were evaluated with Transmission Electron Microscopy and Congo red staining, while the rate of fibril formation at lower concentrations was monitored with Thioflavin T and confirmed with Scanning Electron Microscopy. In order to assess the cytotoxic effect of the non-polymerized peptides, H9C2 cells were incubated with the peptides for 24 hours at 200μg/mL and 100μg/mL and cell death was determined by lactate dehydrogenase release assay. Results Interestingly, sequence alignment on the variable domains of cardiac related light chains revealed the presence of several conserved domains in patients with AL amyloidosis. The chosen peptides were proven to be amyloidogenic suggesting that the variable domains share common amyloidogenic cores. Treatment of H9C2 cells with the peptides at 200μg/mL led to significant reduction in cell viability compared to vehicle treated cells (p<0.001). Two of the peptides deriving from the IGLV6-57 and IGLV3-01 significantly increased cell death at 100μg/mL (p<0.01 and p<0.001 respectively). During the 24h treatment the tested peptides comprised of soluble species and not amyloid fibrils suggesting that monomeric and oligomeric intermediates are highly toxic. Conclusion We discovered five novel amyloidogenic prone regions of cardiac related variable domains that are associated with cellular toxicity and could be exploited for targeted therapeutic interventions. Funding Acknowledgement Type of funding source: None

scholarly journals Ablation of RIP3 protects from dopaminergic neurodegeneration in experimental Parkinson’s disease

2019 ◽  
Vol 10 (11) ◽  
Author(s):  
Pedro A. Dionísio ◽  
Sara R. Oliveira ◽  
Maria M. Gaspar ◽  
Maria J. Gama ◽  
Margarida Castro-Caldas ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Cell Death ◽  
Cultured Cells ◽  
Substantia Nigra Pars Compacta ◽  
Neuronal Cultures ◽  
Interacting Protein ◽  
Dopaminergic Neurodegeneration

Abstract Parkinson’s disease (PD) is driven by dopaminergic neurodegeneration in the substantia nigra pars compacta (SN) and striatum. Although apoptosis is considered the main neurodegenerative mechanism, other cell death pathways may be involved. In this regard, necroptosis is a regulated form of cell death dependent on receptor interacting protein 3 (RIP3), a protein also implicated in apoptosis and inflammation independently of its pro-necroptotic activity. Here, we explored the role of RIP3 genetic deletion in in vivo and in vitro PD models. Firstly, wild-type (Wt) and RIP3 knockout (RIP3ko) mice were injected intraperitoneally with MPTP (40 mg/kg, i.p.), and sacrificed after either 6 or 30 days. RIP3ko protected from dopaminergic neurodegeneration in the SN of MPTP-injected mice, but this effect was independent of necroptosis. In keeping with this, necrostatin-1s (10 mg/kg/day, i.p.) did not afford full neuroprotection. Moreover, MPTP led to DNA fragmentation, caspase-3 activation, lipid peroxidation and BAX expression in Wt mice, in the absence of caspase-8 cleavage, suggesting intrinsic apoptosis. This was mimicked in primary cortical neuronal cultures exposed to the active MPTP metabolite. RIP3 deficiency in cultured cells and in mouse brain abrogated all phenotypes. Curiously, astrogliosis was increased in the striatum of MPTP-injected Wt mice and further exacerbated in RIP3ko mice. This was accompanied by absence of microgliosis and reposition of glial cell line-derived neurotrophic factor (GDNF) levels in the striata of MPTP-injected RIP3ko mice when compared to MPTP-injected Wt mice, which in turn showed a massive GDNF decrease. RIP3ko primary mixed glial cultures also presented decreased expression of inflammation-related genes upon inflammatory stimulation. These findings hint at possible undescribed non-necroptotic roles for RIP3 in inflammation and MPTP-driven cell death, which can contribute to PD progression.

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