Presence Of Caspase-3 Research Articles

Signal-On Detection of Caspase-3 with Methylene Blue-Loaded Metal-Organic Frameworks as Signal Reporters.

In this work, we report on an electrochemical method for the signal-on detection of caspase-3 and the evaluation of apoptosis based on the biotinylation reaction and the signal amplification of methylene blue (MB)-loaded metal-organic frameworks (MOFs). Zr-based UiO-66-NH2 MOFs were used as the nanocarriers to load electroactive MB molecules. Recombinant hexahistidine (His6)-tagged streptavidin (rSA) was attached to the MOFs through the coordination interaction between the His6 tag in rSA and the metal ions on the surface of the MOFs. The acetylated peptide substrate Ac-GDEVDGGGPPPPC was immobilized on the gold electrode. In the presence of caspase-3, the peptide was specifically cleaved, leading to the release of the Ac-GDEVD sequence. A N-terminal amine group was generated and then biotinylated in the presence of biotin-NHS. Based on the strong interaction between rSA and biotin, rSA@MOF@MB was captured by the biotinylated peptide-modified electrode, producing a significantly amplified electrochemical signal. Caspase-3 was sensitively determined with a linear range from 0.1 to 25 pg/mL and a limit of detection down to 0.04 pg/mL. Further, the active caspase-3 in apoptosis inducer-treated HeLa cells was further quantified by this method. The proposed signal-on biosensor is compatible with the complex biological samples and shows great potential for apoptosis-related diagnosis and the screening of caspase-targeting drugs.

Antioxidant, cytotoxic and apoptotic activities of the rhizome of Zingiber zerumbet Linn. in Ehrlich ascites carcinoma bearing Swiss albino mice

Due to having a long history of traditional uses as a functional food, Zingiber zerumbet was selected here to explore the inherent antioxidant and antineoplastic activities of methanolic extract of its rhizome (MEZZR) against Ehrlich ascites carcinoma (EAC) cells. The rich polyphenol containing MEZZR showed a marked DPPH, ABTS, nitric oxide radicals and lipid peroxidation inhibition activity with an IC50 of 3.43 ± 1.25, 11.38 ± 1.39, 23.12 ± 3.39 and 16.47 ± 1.47 µg/ml, respectively, when compared to the standard catechin. In vivo, MEZZR significantly inhibited EAC cell growth, decreased body weight gain, increased life span and restored the altered hematological characteristics of EAC-bearing mice. Moreover, MEZZR induced nuclear condensation and fragmentation, which are notable features of apoptosis as observed by fluorescence microscopy after staining EAC cells of MEZZR-treated mice with Hoechst 33342. Additionally, in vitro, the cell growth inhibition caused by the MEZZR in MTT assay, was remarkably decreased in the presence of caspase-3, -8 and -9 inhibitors. This study thus suggests that MEZZR may possess promising antiproliferative efficacy against EAC cells by inducing cell apoptosis.

Cucurbit[7]urils induced bimetallic nanoparticles network for ultra-sensitive detection of Caspase-3 based on surface plasmon resonance

A novel signal amplification method for surface plasmon resonance (SPR) was developed to detect Caspase-3 based on cucurbit[7]urils (CB[7]) and bimetallic nanonetwork. Three-dimensional (3D) bimetallic nanonetwork was formed through CB[7]-integrated self-assembly of gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs) (AuNPs-CB[7]-AgNPs) by one-pot synthesis method. Caspase-3 was utilized as a model protein and peptide EEAAADEVDFKKAAAC was used as the substrate. In the presence of Caspase-3, peptide was hydrolyzed and exposed phenylalanine at the N-terminus, which could bind to CB[7] through host-guest interaction. The multivalent receptors CB[7] on bimetallic nanonetwork provide a simple strategy to increase the binding affinities between 3D bimetallic nanonetwork and peptide. Thus, AuNPs-CB[7]-AgNPs could attach to the gold chip with ease. Remarkably, due to the strong optical properties and high refractive index of 3D bimetallic nanonetwork, it has a higher SPR signal enhancement than that of only AuNPs or AgNPs. Therefore, a highly sensitive Caspase-3 sensing strategy could be achieved with a low detection limit of 5.39 pg mL−1, providing a new and facile SPR signal amplification method for detection of cell apoptosis.

Six-in-one peptide functionalized upconversion@polydopamine nanoparticle-based ratiometric fluorescence sensing platform for real-time evaluating anticancer efficacy through monitoring caspase-3 activity

• Six-in-one ratiometric fluorescence sensing platform. • Highly sensitive and selective detection of caspase-3. • The anticancer efficacy evaluation of photothermal (PTT)-chemotherapy. • Real-time noninvasive evaluation of tumor response to PTT and chemotherapy of staurosporine (STS). Herein, a six-in-one peptide functionalized upconversion@polydopamine nanoparticle-based ratiometric fluorescence sensing platform (termed as UCNP@PDA@Cy3-pep) has been fabricated for monitoring the anticancer efficacy of photothermal-chemotherapy in real time through detection of caspase-3 activity. In this work, the upconversion nanoparticle (UCNP) core of UCNP@PDA was employed as an internal reference, while the PDA shell of UCNP@PDA was used as an acceptor of fluorescence resonance energy transfer (FRET) system, photothermal therapy (PTT) agent, loading agent of chemical drug and immobilization platform of Cy3 (donor of FRET) labeled peptide (Cy3-pep), which contains a specific caspase-3 substrate (DEVD) and active tumor-targeting motif (PSP). In the presence of caspase-3, the FRET system was broken through the enzymatic cleavage of DEVD, resulting in a recovery of Cy3 fluorescence emission. Under the optimal conditions, the ratio of recovered Cy3 fluorescence intensity with upconversion luminescent (UCL) intensity was linearly dependent on the caspase-3 concentration within the range of 0.5–50 ng mL −1 and the limit of detection (LOD) was calculated as 0.065 ng mL −1 . Using mouse-bearing MG-63 tumor as a model system, the capability of as-proposed UCNP@PDA@Cy3-pep has been successfully demonstrated through a real-time noninvasive evaluation of tumor response to PTT and chemotherapy of staurosporine (STS).

Autophagy Is Involved in Mesenchymal Stem Cell Death in Coculture with Chondrocytes.

ObjectiveCartilage formation is stimulated in mixtures of chondrocytes and human adipose–derived mesenchymal stromal cells (MSCs) both in vitro and in vivo. During coculture, human MSCs perish. The goal of this study is to elucidate the mechanism by which adipose tissue–derived MSC cell death occurs in the presence of chondrocytes.MethodsHuman primary chondrocytes were cocultured with human MSCs derived from 3 donors. The cells were cultured in monoculture or coculture (20% chondrocytes and 80% MSCs) in pellets (200,000 cells/pellet) for 7 days in chondrocyte proliferation media in hypoxia (2% O2). RNA sequencing was performed to assess for differences in gene expression between monocultures or coculture. Immune fluorescence assays were performed to determine the presence of caspase-3, LC3B, and P62.ResultsRNA sequencing revealed significant upregulation of >90 genes in the 3 cocultures when compared with monocultures. STRING analysis showed interconnections between >50 of these genes. Remarkably, 75% of these genes play a role in cell death pathways such as apoptosis and autophagy. Immunofluorescence shows a clear upregulation of the autophagic machinery with no substantial activation of the apoptotic pathway.ConclusionIn cocultures of human MSCs with primary chondrocytes, autophagy is involved in the disappearance of MSCs. We propose that this sacrificial cell death may contribute to the trophic effects of MSCs on cartilage formation.

Hypochlorous acid reversibly inhibits caspase-3: a potential regulator of apoptosis

Caspase-3 is involved in apoptosis. Here, we examine whether hypochlorous acid (HOCl), a final product of myeloperoxidase (MPO), is a modulator of caspase-3 at relatively low concentrations and also its application on metaphase II mouse oocytes. We utilised caspase-3 activity assay, TUNEL assay, the CellEvent caspase 3/7 fluorescent assay, and the MPO/hydrogen peroxide (H2O2) system on mouse oocytes with and without cumulus cells to examine whether low concentrations of HOCl mediate apoptosis by inhibition of caspase-3. A UV-visible spectrophotometer was used to study caspase-3 activity. To determine whether HOCl mediates apoptosis in mouse oocytes, two different concentrations (10 and 100 µM) of HOCl generated by the MPO/H2O2 system were used as treatments (10 µM had little effect on oocyte quality, while 100 µM showed significant deterioration). Induction of apoptotic cell death was determined by TUNEL Assay and the CellEvent caspase 3/7. HOCl mediates caspase-3 inactivation in a dose dependent manner. Subsequent addition of dithiothreitol caused recovery of caspase-3 activity indicating involvement of the oxidation of the Cys-thiol group. Accumulation of HOCl generated by MPO in the presence of caspase-3 also inhibits MPO but requires higher HOCl concentrations, indicating specificity of lower HOCl concentrations to inhibition of caspase-3. Exposure of oocytes to lower HOCl concentrations generated by MPO-H2O2 system prevents MPO-mediated apoptosis whereas exposure to higher HOCl (100 µM) showed apoptosis. Similar results were observed by using the CellEvent caspase 3/7 assay. Low concentrations of HOCl inhibit caspase-3 activity, and may play a role in regulating apoptosis, thus affecting oocyte quality.HighlightsCaspase-3 is involved in apoptosis pathway and loss of this regulation is seen in several diseases.These conditions are associated with inflammation and higher myeloperoxidase (MPO) activity.We examined whether hypochlorous acid (HOCl), generated by MPO, is a modulator of caspase-3.Caspase-3 activity showed a dose dependent decrease with HOCl and this reaction was reversible.HOCl modulates caspase-3 activity and may play a physiological role in regulating apoptosis.

Photoelectrochemical Approach to Apoptosis Evaluation via Multi-Functional Peptide- and Electrostatic Attraction-Guided Excitonic Response.

The excitonic response between nanomaterials is distance-dependent, and thus, interparticle distance is a key factor in fabricating diverse photoelectrochemical (PEC) systems. Current studies focus on DNA-mediated regulation of interparticle distance. However, limited by high demands of base-pairing and flexibility of DNA, it is hard for DNA to achieve precise regulation, especially in a short distance. To pursue better PEC performances in bioanalyses, alternative biological materials should be explored to replace DNA as new "distance controllers". In this work, a peptide with three functional sequences is designed to control interparticle distance between positive-charged Au nanoparticles ((+) AuNPs) and negative-charged CdTe quantum dots ((-) CdTe QDs). Relying on the function of binding sequence, (+) AuNPs and (-) CdTe QDs may be separated to a certain distance by the multifunctional peptide. In this case, the excitonic response is relatively weak, and an evident PEC response can be observed. Because it contains the substrate sequence of caspase-3, the peptide is cleaved in the presence of caspase-3. As a result, without the support of intact peptide, electrostatic attraction plays a dominant role, leading to the aggregation of oppositely charged AuNPs and CdTe QDs, which strengthens the excitonic response and attenuates the PEC response. On the basis of these principles, a novel PEC approach was fabricated to sensitively quantify caspase-3. Meanwhile, caspase-3 in staurosporine-treated A549 cells are also determined by the approach, and the obtained results agree well with the fluorescent intensity of confocal images, manifesting that the proposed PEC method can monitor apoptosis in a label-free strategy. Overall, the study reveals the capability of peptides in controlling interparticle distance of nanomaterials, which may accelerate the development of peptide-based PEC analytical methods.

Guests involved CB[8] capped silver nanoparticles as a means of electrochemical signal enhancement for sensitive detection of Caspase-3

Cysteine aspartase protease (Caspase-3) plays an important role in various diseases. In this work, silver nanoparticles (AgNPs) with electronic properties have been utilized as signal amplification elements for fabricating electrochemical Caspase-3 biosensor. In the presence of Caspase-3, the cleavage of substrate results in the exposure of a free phenylalanine at the end of the peptide, which can bind CB[8] specifically through host-guest reaction. To enhance the electrochemical signal, AgNPs further reacts with CB[8]. Through layer-by-layer assembly of AgNPs, nanostructure was formed via electrostatic cross-linking of AgNPs and CB[8], which can not only prevent the AgNPs’ agglomeration, but also enhance the electrochemical signals. Furthermore, different guests (methylviologen and anthracene) were studied for its electrical transfer efficiency between electrode and AgNPs redox center. Among them, it was found methylviologen/CB[8] complex can increase the electrical conductivity and thus further enhance the Caspase-3 sensor’s sensitivity. Under the optimized detection condition, the detection limit for Caspase-3 is 24.62 pg mL−1. Attribute to its simple and sensitive properties, the fabricated Caspase-3 electrochemical sensor has great potential in clinical applications.

Blocking/Knocking out Smad3 Alleviates Doxorubicin Effects on Endothelial Cells

Recent studies have shown that patients with early-stage cancers are now more likely to die from complications of anticancer therapy than cancer. Doxorubicin (Dox), a drug used to treat numerous cancers, is known to cause delayed cardiomyopathy. We have previously reported reduced microvascular density and increased levels of TGFbeta related transcripts in Dox treated mouse hearts. We hypothesized that microvascular remodeling in Dox treated hearts is mediated by enhanced TGFbeta/Smad3 signaling. We observed the inhibition of human umbilical vein endothelial cell (HUVEC) proliferation and suppression of the formation of vessel-like structures by Dox. TGFbeta pathway inhibitor SB431542 (SB), which selectively blocks Smad2/3 signaling, alleviated the effects of Dox in vitro. In aortic ring experiments, Dox decreased the number of sprouts in aortas from wild type mice while aortic preparations from Smad3 knockout animals were more resistant to the deleterious effect of Dox and produced increased numbers of sprouts. In experiments with co-cultures of cardiac fibroblasts and HUVEC, we identified the detachment of endothelial cells as an additional putative mechanism of microvascular remodeling by Dox. Live imaging of co-cultures treated with Dox in the presence of caspase-3/7 substrate revealed that detaching endothelial cells were not undergoing apoptosis prior to detachment. SB prevented detachment and increased the number of endothelial cells in co-cultures treated with Dox. Treatment with Dox led to the reduced adhesion of endothelial cells to laminin, a major constituent of the endothelial basement membrane while SB improved adhesion of Dox treated cells to laminin. In conclusion, Dox causes inhibition of sprouting angiogenesis in aortic rings and detachment of endothelial cells in an in vitro co-culture model of formation of vessel-like structures. These deleterious processes appear to be mediated by TGFbeta/Smad3 signaling as they were alleviated by both a pathway inhibitor and a genetic Smad3 knockout.

CB[7]-mediated signal amplification approach for sensitive surface plasmon resonance spectroscopy

Cucurbit[7]uril (CB[7]) has received increasing attention because of its unique structure and multiple recognition properties. To improve the sensitivity of surface plasmon resonance (SPR) biosensors, we designed a novel strategy in which caspase-3 serves as the model analyte and CB[7]-modified AuNPs (CB[7]-AuNPs) act as the intermedium. The substrate peptides can be cleaved and replaced with a new N-terminal Phe residue in presence of caspase-3. The CB[7]-AuNPs combine with the N-terminal Phe on the gold chip surface through incorporating the side chain within the nonpolar CB[7] cavity and chelating the N-terminal ammonium group with CB[7] carbonyl oxygen. Then CB[7]-AuNPs integrate with short peptide-modified AuNPs containing Phe at the N-terminal of the peptide. SPR signals are significantly improved through the layer-by-layer assembly of AuNPs. The well-designed sensing platform allows the detection of caspase-3 in a linear range from 10fg/mL to 103fg/mL with a detection limit of 2.2 fg/mL. Given its high specificity and desirable sensitivity, this CB[7]-assisted SPR method may be a useful tool for the assay of caspase-3 in the future. This work may also afford a new model to improve the sensitivity and selectivity of SPR biosensors in other protein detection experiments and disease diagnosis.

Solanum tuberosum lectin inhibits Ehrlich ascites carcinoma cells growth by inducing apoptosis and G2/M cell cycle arrest.

Recently, a lectin was purified from the potato cultivated in Bangladesh locally known as Sheel. In the present study cytotoxicity of the lectin against Ehrlich ascites carcinoma (EAC) cells was studied by MTT assay in vitro in RPMI-1640 medium and 8.0-36.0% cell growth inhibition was observed at the range of 2.5-160μg/ml protein concentration when incubated for 24h. The lectin-induced apoptosis in EAC cells was confirmed by fluorescence and optical microscope. The apoptotic cell death was also confirmed by using caspase inhibitors. Cells growth inhibition caused by the lectin (36%) was remarkably decreased to 7.6 and 22.3% respectively in the presence of caspase-3 and -8 inhibitors. RT-PCR was used to evaluate the expression of apoptosis-related genes Bcl-X, p53, and Bax. An intensive expression of Bcl-X gene was observed in untreated control EAC cells with the disappeared of the gene in Sheel-treated EAC cells. At the same time, Bax gene expression appeared only in Sheel-treated EAC cells and the expression level of the p53 gene was increased remarkable after the treatment of EAC cells with the lectin. The lectin showed strong agglutination activity against EAC cells. Flow cytometry was used to study the cell cycle phases of EAC cells and it was observed that the lectin arrested the G2/M phase. In conclusion, Sheel lectin inhibited EAC cells growth by inducing apoptosis.

Cell-in-Cell Death Is Not Restricted by Caspase-3 Deficiency in MCF-7 Cells.

PurposeCell-in-cell structures are created by one living cell entering another homotypic or heterotypic living cell, which usually leads to the death of the internalized cell, specifically through caspase-dependent cell death (emperitosis) or lysosome-dependent cell death (entosis). Although entosis has attracted great attention, its occurrence is controversial, because one cell line used in its study (MCF-7) is deficient in caspase-3.MethodsWe investigated this issue using MCF-7 and A431 cell lines, which often display cell-in-cell invasion, and have different levels of caspase-3 expression. Cell-in-cell death morphology, microstructures, and signaling pathways were compared in the two cell lines.ResultsOur results confirmed that MCF-7 cells are caspase-3 deficient with a partial deletion in the CASP-3 gene. These cells underwent cell death that lacked typical apoptotic properties after staurosporine treatment, whereas caspase-3-sufficient A431 cells displayed typical apoptosis. The presence of caspase-3 was related neither to the lysosome-dependent nor to the caspase-dependent cell-in-cell death pathway. However, the existence of caspase-3 was associated with a switch from lysosome-dependent cell-in-cell death to the apoptotic cell-in-cell death pathway during entosis. Moreover, cellular hypoxia, mitochondrial swelling, release of cytochrome C, and autophagy were observed in internalized cells during entosis.ConclusionThe occurrence of caspase-independent entosis is not a cell-specific process. In addition, entosis actually represents a cellular self-repair system, functioning through autophagy, to degrade damaged mitochondria resulting from cellular hypoxia in cell-in-cell structures. However, sustained autophagy-associated signal activation, without reduction in cellular hypoxia, eventually leads to lysosome-dependent intracellular cell death.

Optimization of a Stable Linker Involved DEVD Peptide-Doxorubicin Conjugate That Is Activated upon Radiation-Induced Caspase-3-Mediated Apoptosis.

The current study demonstrates the process of selecting an optimal structure for a caspase-3-cleavable doxorubicin prodrug that could be synthesized by simple chemistry in high yields. The prodrug was intended to activate in the presence of caspase-3, whose expression can be exogenously regulated by inducing apoptosis with radiation therapy at a specific site of interest. For this purpose, doxorubicin was conjugated with a DEVD peptide via a heterobifunctional linker. Since the active form of the prodrug comprises the linker besides doxorubicin, we tested several different linkers and selected EMCS based on the examination of its in vitro biological activities. Consequently, DEVD-cysteamide-EMCS-doxorubicin was synthesized as the final compound. According to the various in vitro and in vivo studies, the synthesized prodrug was highly selective for tumors when coupled with radiation therapy, with the added benefit of ease of production.

Expression of executioner procaspases and their activation by a procaspase-activating compound in chronic lymphocytic leukemia cells

AbstractIntrinsic and extrinsic apoptotic pathways converge to activate common downstream executioner caspases (caspase-3, -6, and -7), resulting in cell death. In chronic lymphocytic leukemia (CLL), neoplastic B cells evade apoptosis owing to the overexpression of survival proteins. We hypothesized that direct activation of procaspases could bypass the apoptosis resistance induced by the upstream prosurvival proteins. The procaspase-activating compounds (PAC-1), including B-PAC-1 (L14R8), convert inactive executioner procaspases to their active cleaved forms by chelation of labile zinc ions. Both at transcript and protein levels, primary CLL cells express high levels of latent procaspases (3, -7, and -9). B-PAC-1 treatment induced CLL lymphocyte death which was higher than that in normal peripheral blood mononuclear cells or B cells, and was independent of prognostic markers and microenvironmental factors. Mechanistically, B-PAC-1 treatment activated executioner procaspases and not other Zn-dependent enzymes. Exogenous zinc completely, and pancaspase inhibitors partially, reversed B-PAC-1–induced apoptosis, elucidating the zinc-mediated mechanism of action. The cell demise relied on the presence of caspase-3/7 but not caspase-8 or Bax/Bak proteins. B-PAC-1 in combination with an inhibitor of apoptosis protein antagonist (Smac066) synergistically induced apoptosis in CLL samples. Our investigations demonstrated that direct activation of executioner procaspases via B-PAC-1 treatment bypasses apoptosis resistance and is a novel approach for CLL therapeutics.

Caspase-9 mediates Puma activation in UCN-01-induced apoptosis.

The protein kinase inhibitor 7-hydroxystaurosporine (UCN-01) is one of the most potent and frequently used proapoptotic stimuli. The BH3-only molecule of Bcl-2 family proteins has been reported to contribute to UCN-01-induced apoptosis. Here we have found that UCN-01 triggers Puma-induced mitochondrial apoptosis pathway. Our data confirmed that Akt-FoxO3a pathway mediated Puma activation. Importantly, we elucidate the detailed mechanisms of Puma-induced apoptosis. Our data have also demonstrated that caspase-9 is a decisive molecule of Puma induction after UCN-01 treatment. Caspase-9 mediates apoptosis through two kinds of feedback loops. On the one hand, caspase-9 enhances Puma activation by cleaving Bcl-2 and Bcl-xL independent of caspase-3. On the other hand, caspase-9 directly activated caspase-3 in the presence of caspase-3. Caspase-3 could cleave XIAP in an another positive feedback loop to further sensitize cancer cells to UCN-01-induced apoptosis. Therefore, caspase-9 mediates Puma activation to determine the threshold for overcoming chemoresistance in cancer cells.

Light-up bioprobe with aggregation-induced emission characteristics for real-time apoptosis imaging in target cancer cells.

Specific bioprobes that are capable of real-time and targeted monitoring and imaging of cancer cell apoptosis are highly desirable for cancer diagnosis and the evaluation of cancer therapy efficacy. In this work, an asymmetric fluorescent light-up bioprobe with aggregation-induced emission (AIE) characteristics was designed and synthesized by the conjugation of two different hydrophilic peptides, caspase-specific Asp-Glu-Val-Asp (DEVD) and cyclic Arg-Gly-Asp (cRGD), onto a typical AIE luminogen of a tetraphenylsilole (TPS) unit. The asymmetric probe is almost non-emissive in aqueous solution and its fluorescence is significantly switched on in the presence of caspase-3. The fluorescence turn-on is due to the cleavage of the DEVD moiety by caspase-3, and the aggregation of released TPS-cRGD residues, which restricts the intramolecular rotations of TPS phenyl rings and populates the radiative decay channels. Application of the asymmetric light-up probe for real-time targeted imaging of cancer cell apoptosis is successfully demonstrated using integrin αvβ3 receptor overexpressing U87MG human glioblastoma cells as an example. The probe shows specific targeting capability to U87MG cancer cells by virtue of the efficient binding between cRGD and integrin αvβ3 receptors and is able to real-time monitor and image cancer cell apoptosis in a specific and sensitive manner.

Copper(II) mixed chelate compounds induce apoptosis through reactive oxygen species in neuroblastoma cell line CHP-212

In the present work we report the antiproliferative activity of Cu(II) coordination compounds, CasIIgly ([Cu(4,7-dimethyl-1,10-phenanthroline) (glycinato) (H2O)]NO3), CasIIIia ([Cu(4,4′-dimethyl-2,2′-bipyridine) (glycinato) (H2O)]NO3), and CasIIIEa ([Cu(4,7-dimethyl-1,10-phenanthroline) (acetylacetonato) (H2O)]NO3), against human tumoral cell line CHP-212 (estromal neuroblastoma). Additionally, the molecular structure of CasIIIEa was reported. The IC50 values obtained for the evaluated compounds are in the range 18 to 47μM, representing an inhibition potency increase of 5 to 12 times compared with cisplatin (IC50=226.7μM). After 2h of incubation with the evaluated compounds, cells showed high levels of reactive oxygen species and a considerable GSH depletion, besides an important disruption of the mitochondrial membrane with release of cytochrome C and besides the presence of caspase-3, an effector caspase that is activated in the last step of apoptosis cascade. The results confirm that cell death in neuroblastoma CHP-212 treated with Casiopeínas occurs via apoptosis. Due to the lack of expression of caspase-8, cell death is principally by the mitochondrial pathway. Thus, one of the most interesting findings of this work is the identification of a very important damage in neuroblastoma cells induced by Cu(II) coordination compounds in a very short exposition times.

Agaritine from Agaricus blazei Murrill induces apoptosis in the leukemic cell line U937

Background Agaricus blazei Murrill (ABM) has been shown to exhibit immunostimulatory and anti-cancer activities; however, its mechanism of action is poorly understood. We recently found that the diffusible fraction of hot-water extract of ABM exhibits anti-tumor activity toward leukemic cells, and identified it as agaritine, a hydrazine-containing compound. In the present study, we examined the morphological and cytochemical effects of agaritine on U937 cells to elucidate the tumoricidal mechanism of agaritine. Methods Surface expression of phosphatidylserine (evaluated by annexin V binding), Fas antigen, DNA cleavage using TUNEL staining, changes in caspase activities and cytochrome c release, before and after treatment with agaritine, were examined using U937 cells. Results Nuclear damage, DNA fragmentation, was observed by Wright–Giemsa, TUNEL staining and agarose gel electrophoresis when U937 cells were incubated with 10 μg/mL of agaritine for 48 h. Flow cytometric analysis indicated that agaritine augments the proportion of annexin V-positive U937 cells without significant change in Fas antigen expression. Activities of caspase-3, -8 and -9 were gradually increased after the addition of agaritine. In the presence of caspase-3 or granzyme B inhibitor, except for the caspase-8 inhibitor, annexin V expression was significantly decreased, suggesting that mainly caspase-3 and -9 participate in the apoptotic pathway. Furthermore, cytochrome c release was detected by western blotting analysis after agaritine treatment. Conclusions These results strongly suggest that the ABM constituent agaritine moderately induces apoptosis in U937 leukemic cells via caspase activation through cytochrome c release from mitochondria. General significance This is the first report suggesting that the anti-tumor effect of agaritine is mediated through apoptosis. The present results might provide helpful suggestions for the design of anti-tumor drugs toward leukemia patients.

The N-terminal Fragment from Caspase-cleaved Serine/Arginine Protein-specific Kinase2 (SRPK2) Translocates into the Nucleus and Promotes Apoptosis

SRPK2 belongs to a family of serine/arginine (SR) protein-specific kinases (SRPKs), which phosphorylate SR domain-containing proteins in the nuclear speckles and mediate the pre-mRNA splicing. Previous studies have shown that SRPK2 plays a pivotal role in cell proliferation and apoptosis. However, how SRPK2 is regulated during the apoptosis is unclear. Here, we show that SRPK2 is cleaved by caspases at Asp-139 and -403 residues. Its N terminus cleaved product translocates into the nucleus and promotes VP16-induced apoptosis. Akt phosphorylation of SRPK2 prevents its apoptotic cleavage by caspases. 14-3-3β, the binding partner of Akt-phosphorylated SRPK2, further protects it from degradation. Hence, our results suggest that the N-terminal domain of SRPK2 cleaved by caspases translocates into the nucleus, where it promotes chromatin condensation and apoptotic cell death.

Reconstitution of caspase-3 sensitizes MCF-7 breast cancer cells to radiation therapy

Caspase-3 plays an important role in apoptotic execution. Caspase-3 deficiency or down-regulation has been reported in breast and other kinds of cancers. Given the redundancy of caspase cascade, however, the impact of caspase-3 deficiency/down-regulation on radiation-induced apoptosis remains to be defined. In this report, the specific role of caspase-3 in radiotherapy-induced apoptosis was studied using MCF-7 control (MCF-7/pv, caspase-3 deficient) and caspase-3 reconstituted MCF-7 (MCF-7/c3) breast cancer cells. Caspase-3 reconstitution significantly enhanced radiation-induced apoptosis, with a decrease in the survival fraction, an increase in caspase activation, cleavage of cellular death substrates and mitochondrial depolarization. We also found that the activation of caspase-7 was caspase-3-dependent in radiation-induced apoptosis, which suggests a mini-cascade among the effector caspases and that caspase-3 is essential for signal amplification. In comparing the patterns of death substrates cleavage in radiation-induced apoptosis with that in doxorubicin and TNF-alpha-induced apoptosis, we found that cleavage of lamin B and beta-actin was relatively more susceptible to radiation, which is enhanced in the presence of caspase-3, suggesting cytoskeleton proteins might be preferred markers for radiation-induced apoptosis. These data indicate that caspase-3 plays a critical role in radiotherapy-induced apoptosis, and suggest that caspase-3 deficiency may contribute to the radioresistance of breast cancers.