RESUMEN
Enhanced oxidative stress plays a central role in promoting endothelial dysfunction, leading to the development of atherosclerosis. In this study, we investigated the protective effects of the hydrolysates derived from blue mussel (Mytilus edulis) against H2O2-mediated oxidative injury in human umbilical vein endothelial cells (HUVECs). The blue mussel hydrolysates were prepared by enzymatic hydrolysis with eight proteases, and blue mussel-α-chymotrypsin hydrolysate (BMCH) showed the highest antioxidant activities in DPPH radical scavenging, ABTS⺠radical scavenging, and ORAC value compared to those of the other hydrolysates. BMCH also inhibited Cu2+-mediated low density lipoprotein (LDL) oxidation. Treatment of H2O2 resulted in the decreased HUVEC viability whereas pre-treatment with BMCH increased HUVEC viability and reduced reactive oxygen species (ROS) generation. BMCH pre-treatment increased cellular antioxidant capacities, including levels of glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) against H2O2-mediated oxidative stress in HUVECs. Flow cytometry and western blot analysis revealed that BMCH pre-treatment significantly reduced H2O2-mediated HUVEC apoptosis through inhibition of caspase-3 activation. Real-time-qPCR analysis showed that BMCH down-regulated expression of p53 and caspase-3 genes, as well as decreased the bax/bcl-2 ratio. Taken together, these results indicate that BMCH may be useful as functional food ingredients for protecting endothelial dysfunction or related disease.
Asunto(s)
Aminoácidos/química , Caspasa 3/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Mytilus edulis/química , Estrés Oxidativo/efectos de los fármacos , Hidrolisados de Proteína/farmacología , Aminoácidos/metabolismo , Aminoácidos/farmacología , Animales , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Glutatión/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Peróxido de Hidrógeno/administración & dosificación , Lipoproteínas LDL/metabolismo , Mytilus edulis/metabolismo , Hidrolisados de Proteína/química , Hidrolisados de Proteína/aislamiento & purificación , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Cadmium (Cd) is a highly toxic environmental pollutant released from the smelting and refining of metals and cigarette smoking. Oral exposure to cadmium may result in adverse effects on a number of tissues, including the central nervous system (CNS). In fact, its toxicity has been related to neurological disorders, as well as neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Under normal conditions, Cd barely reaches the brain in adults because of the presence of the blood-brain barrier (BBB); however, it has been demonstrated that Cd-dependent BBB alteration contributes to pathogenesis of neurodegeneration. However, the mechanism underlying Cd-dependent BBB alteration remain obscure. Here, we investigated the signaling pathway of Cd-induced tight junction (TJ), F-actin, and vimentin protein disassembly in a rat brain endothelial cell line (RBE4). RBE4 cells treated with 10 µM cadmium chloride (CdCl2) showed a dose- and time-dependent significant increase in reactive oxygen species (ROS) production. This phenomenon was coincident with the alteration of the TJ zonula occludens-1 (ZO-1), F-actin, and vimentin proteins. The Cd-dependent ROS increase elicited the upregulation of GRP78 expression levels, a chaperone involved in endoplasmic reticulum (ER) stress that induces caspase-3 activation. Further signal profiling by the pannexin-1 (PANX1) specific inhibitor 10Panx revealed a PANX1-independent increase in ATP spillage in Cd-treated endothelial cells. Our results point out that a ROS-dependent ER stress-mediated signaling pathway involving caspase-3 activation and ATP release is behind the BBB morphological alterations induced by Cd.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Cadmio/metabolismo , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/metabolismo , Actinas/metabolismo , Animales , Barrera Hematoencefálica/citología , Línea Celular , Estrés del Retículo Endoplásmico , Células Endoteliales/citología , Células Endoteliales/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Vimentina/metabolismoRESUMEN
In this study, our aim was to determine whether caspase 3 plays a role, during previtellogenesis, in the ovarian follicular epithelium of the lizard Podarcis sicula. We investigated the presence and localization of proform and active caspase 3 by enzyme assay, Western blotting and immunocytochemistry. In parallel, a fragment of caspase 3 was cloned for the first time in this species, sequenced and used for in situ hybridization to localize messengers and analysed by a phylogenetic survey to shed light on its homology with reptilian caspases. Results demonstrated that: (1) the follicle cells expressed a caspase of the 3/7 group and the mRNA for caspase 3 was transcribed in the stem phase and was completely translated during cell differentiation; (2) the proform protein was stored during the differentiated (nurse) stage and activated at the end of previtellogenesis provoking the degeneration of cells; (3) the predicted protein sequence, although partial, had a strong similarity with the known reptilian caspases 3. The epithelial cells of the ovarian follicle, therefore, do not employ caspase 3 during the nurse stage but, instead, prepare for apoptosis long before the process actually begins. The relevance of this strategy is discussed.
Asunto(s)
Caspasa 3/metabolismo , Lagartos/metabolismo , Folículo Ovárico/citología , Folículo Ovárico/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Caspasa 3/química , Caspasa 3/genética , Caspasa 7/metabolismo , Clonación Molecular , Pruebas de Enzimas , Femenino , Regulación Enzimológica de la Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Ninety samples from the extracts of plants from traditional Chinese medicines were screened for antitumor activity. Paeoniflorigenone (PFG) was isolated as an active ingredient from the root of moutan cortex, which showed the strongest activity. In addition, our data indicated that PFG was cytotoxic and induced apoptosis selectively in the cancer cell lines. These effects were cancelled by the addition of caspase inhibitor Z-VAD-FMK, suggesting that it was mediated by caspase-3 activation.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Citotoxinas/farmacología , Medicamentos Herbarios Chinos/química , Monoterpenos/farmacología , Paeonia/química , Células 3T3-L1 , Clorometilcetonas de Aminoácidos/antagonistas & inhibidores , Clorometilcetonas de Aminoácidos/farmacología , Animales , Antineoplásicos Fitogénicos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Apoptosis/genética , Caspasa 3/genética , Caspasa 3/metabolismo , Inhibidores de Caspasas/farmacología , Proliferación Celular/efectos de los fármacos , Citotoxinas/aislamiento & purificación , Fragmentación del ADN/efectos de los fármacos , Expresión Génica , Células HL-60 , Células HeLa , Humanos , Células Jurkat , Medicina Tradicional China , Ratones , Monoterpenos/aislamiento & purificación , Extractos Vegetales/química , Raíces de Plantas/químicaRESUMEN
BRCA1/2-mutant cells are hypersensitive to inactivation of poly(ADP-ribose) polymerase 1 (PARP-1). We recently showed that inhibition of PARP-1 by NU1025 is strongly cytotoxic for BRCA1-positive BT-20 cells, but not BRCA1-deficient SKBr-3 cells. These results raised the possibility that other PARP-1 inhibitors, particularly those tested in clinical trials, may be more efficacious against BRCA1-deficient SKBr-3 breast cancer cells than NU1025. Thus, in the presented study the cytotoxicity of four PARP inhibitors under clinical evaluation (olaparib, rucaparib, iniparib and AZD2461) was examined and compared to that of NU1025. The sensitivity of breast cancer cells to the PARP-1 inhibition strongly varied. Remarkably, BRCA-1-deficient SKBr-3 cells were almost completely insensitive to NU1025, olaparib and rucaparib, whereas BRCA1-expressing BT-20 cells were strongly affected by NU1025 even at low doses. In contrast, iniparib and AZD2461 were cytotoxic for both BT-20 and SKBr-3 cells. Of the four tested PARP-1 inhibitors only AZD2461 strongly affected cell cycle progression. Interestingly, the anti-proliferative and pro-apoptotic potential of the tested PARP-1 inhibitors clearly correlated with their capacity to damage DNA. Further analyses revealed that proteomic signatures of the two studied breast cancer cell lines strongly differ, and a set of 197 proteins was differentially expressed in NU1025-treated BT-20 cancer cells. These results indicate that BT-20 cells may harbor an unknown defect in DNA repair pathway(s) rendering them sensitive to PARP-1 inhibition. They also imply that therapeutic applicability of PARP-1 inhibitors is not limited to BRCA mutation carriers but can be extended to patients harboring deficiencies in other components of the pathway(s).
Asunto(s)
Proteína BRCA1/genética , Neoplasias de la Mama/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Ftalazinas/administración & dosificación , Piperidinas/administración & dosificación , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificación , Poli(ADP-Ribosa) Polimerasas/biosíntesis , Quinazolinas/administración & dosificación , Apoptosis/efectos de los fármacos , Proteína BRCA1/biosíntesis , Benzamidas/administración & dosificación , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Indoles/administración & dosificación , Piperazinas/administración & dosificación , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/genéticaRESUMEN
A series of terphenyl based 4-aza-2,3-didehydropodophyllotoxin conjugates (8a-r) were synthesized by a straightforward one-step multicomponent synthesis that demonstrated anticancer activity against five human cancer cell lines (lung, colon, renal, prostate and cervical). All the tested compounds showed potent anticancer activity with IC50 values ranging from 0.87 to 16.59 µM. Among them compounds 8n and 8p showed significant anticancer activity in lung cancer cells with IC50 values 0.91 and 0.87 µM, respectively. Flow cytometric analysis revealed that these compounds induced cell cycle arrest in G2/M phase in A549 cell line leading to caspase-3 dependent apoptotic cell death. The tubulin polymerization assay and immunofluorescence analysis showed that these compounds effectively inhibit microtubule assembly at both molecular and cellular levels in A549 cells. Further, Hoechst staining, DNA fragmentation analysis also suggested that these compounds induced cell death by apoptosis. Overall, the current study demonstrated that the synthesis of terphenyl based 4-aza-2,3-didehydropodophyllotoxin conjugates as promising anticancer agents with G2/M cell cycle arrest and apoptotic-inducing activities via targeting tubulin.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/síntesis química , Podofilotoxina/análogos & derivados , Moduladores de Tubulina/síntesis química , Tubulina (Proteína)/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular Tumoral , Fragmentación del ADN/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Podofilotoxina/síntesis química , Podofilotoxina/química , Podofilotoxina/farmacología , Polimerizacion/efectos de los fármacos , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologíaRESUMEN
1,3,4-Thiadiazoles are structures that are bioisosteres of 1,3,4-oxadiazole and pyrimidine ring, which are found in the structure of many drugs and anticancer active newly studied derivatives. In the past, high effect profiles have been observed in many molecules created, based on the anticancer effects of the 2-amino-1,3,4-thiadiazole (NSC 4728) molecule and acetazolamide molecules. Focusing on these molecules and evaluating them in terms of mechanistic effects, twelve new N-[5-((3,5-dichlorophenoxy) methyl]-1,3,4-thiadiazole derivatives (3a-3i) were synthesized and their biological activities were investigated in lung cancer cells. The anticancer effects of the compounds were evaluated on the A549 and L929 cell lines. Compound 3f, namely 2-[(5-chlorobenzotiyazol-2-yl)thio]-N-[5-[(3,5-dichlorophenoxy)methyl]-1,3,4-thiadiazol-2-yl]acetamide, showed better activity than cisplatin, exhibiting high inhibitory potency (IC50: <0.98 µg/mL) and selectivity against A549 cell line even at the lowest concentration tested. Compounds 3c, 3f, and 3h with the lowest IC50 values of the compounds exhibited an excellent percentage of apoptosis between 72.48 and 91.95% compared to cisplatin. The caspase-3 activation and mitochondrial membrane potential change of the aforementioned three compounds were also studied. Moreover, matrix metalloproteinase-9 (MMP-9) inhibition potential of all final compounds was also investigated and IC50 values for compounds 3b and 3g were identified as 154.23 and 107.28 µM. Molecular docking and molecular dynamic simulation studies for MMP-9 enzyme inhibition were realized on these compounds and the nitrogen atoms of amide and thiadiazole moieties' ascertained that they play a key role in chelating with Zn metal, at the same time, (thio)ether moieties allow conformational change resulting in the ligand can make more stable contacts.Communicated by Ramaswamy H. Sarma.
RESUMEN
The "click chemistry" approach utilizing 5-ethynyl-2'-deoxyuridine (EdU) as a DNA precursor was recently introduced to assess DNA replication and adapted to flow- and imaging-cytometry. In the present study, we observed that EdU, once incorporated into DNA, induces DNA damage signaling (DDS) such as phosphorylation of ATM on Ser1981, of histone H2AX on Ser139, of p53 on Ser15, and of Chk2 on Thr68. It also perturbs progression of cells through the cell cycle and subsequently induces apoptosis. These effects were observed in non-small cell lung adenocarcinoma A549 as well as in B-cell human lymphoblastoid TK6 and WTK1 cells, differing in the status of p53 (wt versus mutated). After 1 h EdU pulse-labeling, the most affected was cells progression through the S phase subsequent to that at which they had incorporated EdU. This indicates that DNA replication using the template containing incorporated EdU is protracted and triggers DDS. Furthermore, progression of cells having DNA pulse-labeled with EdU led to accumulation of cells in G2 , likely by activating G2 checkpoint. Consistent with the latter was activation of p53 and Chk2. Although a correlation was observed in A549 cells between the degree of EdU incorporation and the extent of γH2AX induction, such correlation was weak in TK6 and WTK1 cells. The degree of perturbation of the cell cycle kinetics by the incorporated EdU was different in the wt p53 TK6 cells as compared to their sister WTK1 cell line having mutated p53. The data are thus consistent with the role of p53 in modulating activation of cell cycle checkpoints in response to impaired DNA replication. The confocal microscopy analysis of the 3D images of cells exposed to EdU for 1 h pulse and then grown for 24 or 48 h revealed an increased number of colocalized γH2AX and p53BP1 foci considered to be markers of DNA double-strand breaks and enlarged nuclei.
Asunto(s)
Química Clic/métodos , Daño del ADN/genética , ADN/genética , Desoxiuridina/análogos & derivados , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , ADN/efectos de los fármacos , ADN/aislamiento & purificación , Daño del ADN/efectos de los fármacos , Desoxiuridina/química , Histonas/genética , Histonas/aislamiento & purificación , Humanos , Citometría de Barrido por Láser/métodos , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/aislamiento & purificaciónRESUMEN
The lipophilic, cell-penetrating zinc chelator N,N,N',N',-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN, 1) and the zinc chelating procaspase-activating compound PAC-1 (2) both have been reported to induce apoptosis in various cell types. The relationship between apoptosis-inducing ability and zinc affinity (Kd), have been investigated with two new model compounds, ZnA-DPA (3) and ZnA-Pyr (4), and compared to that of TPEN and PAC-1. The zinc-chelating o-hydroxybenzylidene moiety in PAC-1 was replaced with a 2,2'-dipicoylamine (DPA) unit (ZnA-DPA, 3) and a 4-pyridoxyl unit (ZnA-Pyr, 4), rendering an order of zinc affinity TPEN>ZnA-Pyr>ZnA-DPA>PAC-1. The compounds were incubated with the rat pheochromocytoma cell line PC12 and cell death was measured in combination with ZnSO4, a caspase-3 inhibitor, or a ROS scavenger. The model compounds ZnA-DPA (3) and ZnA-Pyr (4) induced cell death at higher concentrations as compared to PAC-1 and TPEN, reflecting differences in lipophilicity and thereby cell-penetrating ability. Addition of ZnSO4 reduced cell death induced by ZnA-Pyr (4) more than for ZnA-DPA (3). The ability to induce cell death could be reversed for all compounds using a caspase-3-inhibitor, and most so for TPEN (1) and ZnA-Pyr (4). Reactive oxygen species (ROS), as monitored using dihydro-rhodamine (DHR), were involved in cell death induced by all compounds. These results indicate that the Zn-chelators ZnA-DPA (3) and ZnA-Pyr (4) exercise their apoptosis-inducing effect by mechanisms similar to TPEN (1) and PAC-1 (2), by chelation of zinc, caspase-3 activation, and ROS production.
Asunto(s)
Quelantes/síntesis química , Etilenodiaminas/química , Hidrazonas/química , Piperazinas/química , Zinc/química , Aminas/química , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/química , Caspasa 3/metabolismo , Inhibidores de Caspasas/síntesis química , Inhibidores de Caspasas/química , Inhibidores de Caspasas/toxicidad , Quelantes/química , Quelantes/toxicidad , Etilenodiaminas/toxicidad , Hidrazonas/toxicidad , Células PC12 , Ácidos Picolínicos/química , Piperazinas/toxicidad , Piridoxina/química , Ratas , Especies Reactivas de Oxígeno/metabolismo , Sulfato de Zinc/química , Sulfato de Zinc/toxicidadRESUMEN
BACKGROUND: The cancer is still a major cause of death worldwide. Among different targets to design anticancer agents, caspase-3 is an important target as its cleavage and activation lead to apoptosis and finally, cancer cell death. Apart from some naturally occurring molecules, many small molecules have been reported as caspase-3 activators. OBJECTIVES: In view of the above, the objective has been to review the published work on small molecules reported as caspase-3 activators and their anticancer activity to get some novel lead molecules for designing novel molecules of improved cancer therapeutic. METHODS: Literature search has been carried out using different search engines like google, Elsevier, Science direct, RSC, etc. for the publications of small molecules as caspase-3 activators inducing apoptosis in cancer cells. RESULTS: In this review, the small molecules showing caspase-3 cleavage and activation have been discussed under different broad chemical classes so as to provide some insight into the structural features responsible for caspase-3 activation leading to anticancer activity. The review also encompasses the established drugs, novel organometallics showing caspase-3 activation and anticancer activity. CONCLUSION: A large number of small molecules including some established drugs and organometallics have shown cleavage and activation of caspase-3 leading to apoptosis and anticancer activity. Many reported potent molecules of different chemical classes may be useful as lead molecules for optimization of anticancer activity as well as they may provide an insight of structural features which may be useful in designing novel caspase-3 activators as anticancer agents for drug development.
Asunto(s)
Antineoplásicos , Neoplasias , Humanos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Antineoplásicos/química , Apoptosis , Caspasa 3/metabolismo , Neoplasias/tratamiento farmacológico , Activación EnzimáticaRESUMEN
(1) The cytotoxicity and antioxidant activity of different fractions as well as the pro-apoptotic activity of saponin fractions from Eryngium planum L. in SKOV-3 was investigated. (2) In screening studies, the cytotoxicity of six fractions on SKOV-3 was examined by LDH and SRB assays. The most active fractions-triterpenoid saponins-were selected for further investigation. To determine the mechanism of saponin fractions' cytotoxicity, their ability to induce apoptosis was examined via Annexin V assay. The effect of the saponin fractions on caspase 3 activity was measured using a Caspase 3 Assay Kit. The expression of 84 apoptosis-related genes was investigated in cancer cells exposed to saponin fractions from the roots. The radical scavenging capacity of different fractions was determined via DPPH assay. (3) The pronounced cytotoxic effects in SKOV-3 were demonstrated by saponin fractions from the leaves and roots. Those saponin fractions were chosen for further investigation. The treatment of cancer cell lines with saponins obtained from the roots provoked a significant increase in apoptotic cells. In the SKOV-3 cells, saponins caused upregulation of pro-apoptotic genes and a decrease in anti-apoptotic genes. The activation of caspase 3 was correlated with an increased DFFA expression level in the treated SKOV-3 cells. The most active fractions were phenolic acids from the shoots and roots. (4) To the best of our knowledge, the current study is the first to demonstrate that the barrigenol-type triterpenoid saponin fraction from the roots of E. planum inhibits SKOV-3 cell proliferation and induces apoptosis, which may be regulated by the expression of genes mostly specific to a mitochondria-related pathway.
RESUMEN
Ligand-based pharmacophore modelling and virtual screening along with in vitro screening were performed as a rational strategy for the identification of novel compounds as apoptosis inducers and anticancer agents from the chemical database. Known apoptosis inducers were selected from the literature for generation of pharmacophore models, which were subjected to validation using Receiver operating characteristic (ROC) and Günere-Henry (GH) scoring methods. Based on highest fitness score of 4680.61, ROC value of 0.872 and GH score of 0.758, pharmacophore model-2 was selected as the best model. Model-2 as 3D search query was searched against the IBS database to find novel compounds as hits. Three hits were selected with a QFIT value more than 82 for in vitro screening as apoptosis inducers and anticancer agents. In vitro anticancer activity was performed using resazurin cell variability assay, and apoptosis inducing activity was determined using caspase-3 activation and annexin-FITC assays. One of the retrieved hit, STOCK5S-44056 demonstrated IC50 value of 23.56 µM in cell variability assay, and had EC50 value of 26.95 µM in caspase-3 activation assay. STOCK5S-44056 also indicated late stage induction of apoptosis in annexin assay. The results of in vitro activity revealed that STOCK5S-44056 has a potential to become anticancer agents.
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Antineoplásicos/química , Apoptosis/efectos de los fármacos , Diseño de Fármacos , Relación Estructura-Actividad Cuantitativa , Animales , Bases de Datos de Compuestos Químicos , Humanos , Ligandos , Modelos MolecularesRESUMEN
Resistance to therapeutic drugs occurs in virtually all types of cancers, and the tolerance to one drug frequently becomes broad therapy resistance; however, the underlying mechanism remains elusive. Combining a whole whole-genome-wide RNA interference screening and an evolutionary drug pressure model with MDA-MB-231 cells, it is found that enhanced protein damage clearance and reduced mitochondrial respiratory activity are responsible for cisplatin resistance. Screening drug-resistant cancer cells and human patient-derived organoids for breast and colon cancers with many anticancer drugs indicates that activation of mitochondrion protein import surveillance system enhances proteasome activity and minimizes caspase activation, leading to broad drug resistance that can be overcome by co-treatment with a proteasome inhibitor, bortezomib. It is further demonstrated that cisplatin and bortezomib encapsulated into nanoparticle further enhance their therapeutic efficacy and alleviate side effects induced by drug combination treatment. These data demonstrate a feasibility for eliminating broad drug resistance by targeting its common mechanism to achieve effective therapy for multiple cancers.
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AIMS: To explore the potential mechanism that the role of the Akt/eNOS/NO pathway in calpain-induced caspase-3 and NF-κB activation during septic apoptosis. MAIN METHODS: Septic rats were stimulated by LPS (8â¯mg/kg, i.p.). Myocardial calpain, caspase-3, NO, TNF-α and IL-1ß levels were detected by ELISA. The levels of Akt/p-Akt, eNOS/p-eNOS, iNOS proteins and number of apoptotic cells were evaluated by immunohistochemistry, western blot and TUNEL method. KEY FINDINGS: Compared with sham, LPS treatment resulted in 4.1-fold and 1.8-fold increases in myocardial calpain activity and caspase-3 activation, respectively, and a significant increase (6.8-fold) in apoptotic cardiomyocytes was observed. The administration of calpain inhibitors (calpain inhibitor-IV, PD150606 and PD151746) showed that p-Akt and p-eNOS protein levels were correlated with the levels of LPS-induced myocardial calpain and caspase-3 activity. In addition, the quantity of p-Akt protein and NO content were markedly attenuated by wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor. Pretreatment with L-NAME, an NOS inhibitor, induced a decrease in p-eNOS proteins and apoptosis in myocardial tissues, while iNOS proteins were strongly increased in septic rats. SIGNIFICANCE: This study suggests that the Akt/eNOS/NO pathway might lead to a novel pharmacological therapy for cardiomyocytes apoptosis in sepsis.
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Calpaína/metabolismo , Caspasa 3/metabolismo , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sepsis/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Lipopolisacáridos/toxicidad , Masculino , Miocardio/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Ratas , Ratas Sprague-Dawley , Sepsis/inducido químicamente , Transducción de Señal/fisiologíaRESUMEN
We aimed at studying the potential mechanisms in the preventive effect of resveratrol on serum deprivation induced caspase 3 activation on non-transformed cells. METHODS: Apoptosis was induced by serum deprivation in primary mouse embryonic fibroblasts. Caspase 3 activation, reactive oxygen species production and depolarization of the mitochondrial membrane were measured by fluorescence methods. The involvement of intracellular receptors and autophagy in the effect of resveratrol were analyzed by using specific agonists and antagonists. The role of autophagy was further examined by Western Blot analysis of its protein markers, LC3-II and p62 as well as by acridine orange staining of acidic vacuoles. RESULTS: We found that neither aromatic hydrocarbon receptors nor estrogen receptors play an important role in the cytoprotective effect of resveratrol. Reactive oxygen species production was not significantly altered by either serum deprivation or resveratrol treatment. In the presence of serum deprivation resveratrol however, induced a significant depolarization in mitochondrial membrane potential. The autophagy inhibitor, chloroquine not only eliminated the preventive effect of resveratrol, but also turned it to deleterious suggesting the prominent role of autophagy induction in the cytoprotective effect. Resveratrol did not alter LC3-II expression, but facilitated p62 degradation in serum deprived cells, suggesting its ability to augment the late phase of autophagy and thus promote the autophagic flux. CONCLUSION: We have demonstrated that resveratrol can protect primary fibroblasts against serum deprivation induced apoptosis by provoking mild mitochondrial stress and consequent up-regulation of autophagic flux.
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Caspasa 3/farmacología , Inhibidores de Caspasas/farmacología , Fibroblastos/efectos de los fármacos , Resveratrol/farmacología , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Caspasa 3/metabolismo , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The mechanism for Myc-induced genetic instability is not well understood. Here we show that sublethal activation of Caspase-3 plays an essential, facilitative role in Myc-induced genomic instability and oncogenic transformation. Overexpression of Myc resulted in increased numbers of chromosome aberrations and γH2AX foci in non-transformed MCF10A human mammary epithelial cells. However, such increases were almost completely eliminated in isogenic cells with CASP3 gene ablation. Furthermore, we show that endonuclease G, an apoptotic nuclease downstream of Caspase-3, is directly responsible for Myc-induced genetic instability. Genetic ablation of either CASP3 or ENDOG prevented Myc-induced oncogenic transformation of MCF10A cells. Taken together, we believe that Caspase-3 plays a critical, unexpected role in mediating Myc-induced genetic instability and transformation in mammalian cells.
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Carcinogénesis , Caspasa 3/metabolismo , Transformación Celular Neoplásica , Inestabilidad Genómica , Proteínas Proto-Oncogénicas c-myc/metabolismo , Línea Celular , Células Epiteliales/fisiología , HumanosRESUMEN
Bisphenols are important chemicals that are widely used in the manufacturing of polycarbonates, epoxy resin and thermal paper, and thus the exposure of humans to these substances has been noted. The purpose of this study was to assess eryptotic changes in human erythrocytes exposed (in vitro) to bisphenol A (BPA) and its selected analogs, i.e.,bisphenol F (BPF), bisphenol S (BPS) and bisphenol AF (BPAF). The erythrocytes were incubated with compounds studied at concentrations ranging from 1 to 250µg/mL for 4, 12 or 24h. The results showed that BPA and its analogs increased cytosolic calcium ions level with the strongest effect noted for BPAF. It has also been revealed that all bisphenols analyzed, and BPAF and BPF in particular increased phosphatidylserine translocation in red blood cells, which confirmed that they exhibited eryptotic potential in this cell type. Furthermore, it was shown that BPA and its analogs caused significant increase in calpain and caspase-3 activities, while the strongest effect was noted for BPAF. BPS, which is the main substituent of bisphenol A in polymers and thermal paper production exhibited similar eryptotic potential to BPA. Eryptotic changes in human erythrocytes were provoked by bisphenols at concentrations, which may influence the human body during occupational exposure or subacute poisoning with these compounds.
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Compuestos de Bencidrilo/toxicidad , Eritrocitos/efectos de los fármacos , Fenoles/toxicidad , Adolescente , Adulto , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Calpaína/metabolismo , Caspasa 3/metabolismo , Eritrocitos/metabolismo , Humanos , Persona de Mediana Edad , Fosfatidilserinas/metabolismo , Adulto JovenRESUMEN
INTRODUCTION: Apoptosis is involved in pathological cell death of a wide range of human diseases. One of the most important biochemical markers of apoptosis is activation of caspase-3. Ability to detect caspase-3 activation early in the pathological process is important for determining the timing for interfering with apoptosis initiation and prevention of cell damage. Techniques allowing detection of caspase-3 activity at a single cell level show increased sensitivity, compared to biochemical assays; therefore, we developed a novel bicistronic caspase-3 sensor vector enabling detection of caspase-3 activity in individual cells. METHODS: We employed green fluorescent protein (GFP) as a reporter for caspase-3 activation in our constructs and assessed the functionality of the generated constructs in transiently transfected Neuro2A and HEK293 cells under basal conditions and following application of okadaic acid (OA) or staurosporine (STS) to induce apoptosis. To ensure responsiveness of the new sensor vector to active caspase-3, we co-transfected the sensor with plasmid(s) overexpressing active caspase-3 and quantified GFP fluorescence using a plate reader. RESULTS: We observed an increase in GFP expression in cells transfected with the new bicistronic caspase-3 sensor in response to both OA and STS. We also showed a significant increase in GFP fluorescence intensity in cells co-expressing the sensor with the plasmid(s) encoding active caspase-3. DISCUSSION: We generated a novel bicistronic caspase-3 sensor vector which relies on a transcription factor/response element system. The obtained sensor combines high sensitivity of the single cell level detection with the possibility of automated quantification.