RESUMEN
iCasp9 suicide gene has been widely used as a promising killing strategy in various cell therapies. However, different cells show significant heterogeneity in response to apoptosis inducer, posing challenges in clinical applications of killing strategy. The cause of the heterogeneity remains elusive so far. Here, by simultaneously monitoring the dynamics of iCasp9 dimerization, Caspase3 activation, and cell fate in single cells, we found that the heterogeneity was mainly due to cell-to-cell variability in initial iCasp9 expression and XIAP/Caspase3 ratio. Moreover, multiple-round drugging cannot increase the killing efficiency. Instead, it will place selective pressure on protein levels, especially on the level of initial iCasp9, leading to drug resistance. We further show this resistance can be largely eliminated by combinatorial drugging with XIAP inhibitor at the end, but not at the beginning, of the multiple-round treatments. Our results unveil the source of cell fate heterogeneity and drug resistance in iCasp9-mediated cell death, which may enlighten better therapeutic strategies for optimized killing.
Asunto(s)
Caspasa 9/farmacología , Muerte Celular/efectos de los fármacos , Caspasa 3/metabolismo , Caspasa 9/química , Caspasa 9/metabolismo , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Medicamentos/efectos de los fármacos , Sinergismo Farmacológico , Células HEK293 , Células HeLa , Humanos , Multimerización de Proteína , Tacrolimus/análogos & derivados , Tacrolimus/farmacología , Factores de Tiempo , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
Hexameric hemoprotein (HTHP) is employed as a scaffold protein for the supramolecular assembly and activation of the apoptotic signalling enzyme caspase-9, using short DNA elements as modular recruitment domains. Caspase-9 assembly and activation on the HTHP platform due to enhanced proximity is followed by combinatorial inhibition at high scaffold concentrations. The DNA recruitment domains allow for reversible switching of the caspase-9 assembly and activity state using short modulatory DNA strands. Tuning of the recruitment domain affinity allows for generating kinetically trapped active enzyme complexes, as well as for dynamic repositioning of caspases over scaffold populations and inhibition using monovalent sink platforms. The conceptual combination of a highly structured multivalent protein platform with modular DNA recruitment domains provides emergent biomimicry properties with advanced levels of control over protein assembly.
Asunto(s)
Caspasa 9/metabolismo , ADN/metabolismo , Caspasa 9/química , ADN/química , Humanos , Cinética , Modelos MolecularesRESUMEN
The cysteine-containing aspartate specific proteinase (caspase) family plays important roles in apoptosis and the maintenance of homeostasis in lampreys. We conducted genomic and functional comparisons of six distinct lamprey caspase groups with human counterparts to determine how these expanded molecules evolved to adapt to the changing caspase-mediated signaling pathways. Our results showed that lineage-specific duplication and rearrangement were responsible for expanding lamprey caspases 3 and 7, whereas caspases 1, 6, 8, and 9 maintained a relatively stable genome and protein structure. Lamprey caspase family molecules displayed various expression patterns and were involved in the innate immune response. Caspase 1 and 7 functioned as a pattern recognition receptor with a broad-spectrum of microbial recognition and bactericidal effect. Additionally, caspases 1 and 7 may induce cell apoptosis in a time- and dose-dependent manner; however, apoptosis was inhibited by caspase inhibitors. Thus, these molecules may reflect the original state of the vertebrates caspase family. Our phylogenetic and functional data provide insights into the evolutionary history of caspases and illustrate their functional characteristics in primitive vertebrates.
Asunto(s)
Apoptosis/genética , Caspasas/genética , Inmunidad Innata , Lampreas/genética , Transducción de Señal/inmunología , Animales , Apoptosis/efectos de los fármacos , Caspasa 1/química , Caspasa 1/genética , Caspasa 1/aislamiento & purificación , Caspasa 1/metabolismo , Caspasa 3/química , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 6/química , Caspasa 6/genética , Caspasa 6/metabolismo , Caspasa 7/química , Caspasa 7/genética , Caspasa 7/aislamiento & purificación , Caspasa 7/metabolismo , Caspasa 8/química , Caspasa 8/genética , Caspasa 8/metabolismo , Caspasa 9/química , Caspasa 9/genética , Caspasa 9/metabolismo , Inhibidores de Caspasas/farmacología , Caspasas/química , Caspasas/aislamiento & purificación , Caspasas/metabolismo , Evolución Molecular , Duplicación de Gen , Reordenamiento Génico , Genoma , Genómica , Células HeLa , Humanos , Inmunidad Innata/genética , Lampreas/crecimiento & desarrollo , Lampreas/inmunología , Lampreas/metabolismo , Filogenia , Proteínas Recombinantes , Alineación de Secuencia , Transducción de Señal/genética , Staphylococcus aureus/efectos de los fármacos , Regulación hacia Arriba , Vibrio/efectos de los fármacosRESUMEN
α-Synuclein (αS) aggregates plays a pivotal role in the pathogenesis of synucleinopathies including Parkinson's Disease. The toxicity of αS aggregates has been broadly studied and variant defects have been reported through which these aggregates lead in cell death. Although cell death through apoptosis pathway has been proposed in many studies, the molecular details underlying in this pathway have not been uncovered. To shed a light on the relationships between αS aggregates and apoptotic cell death, changes in levels and behavior of molecular indicators of the intrinsic apoptotic pathway was investigated in HEK-293T cells overexpressing wild-type α-synuclein and A53T-α-synuclein. Overexpression of both WT-αS and A53T-αS resulted in the increase of caspase-9 activity, and rise in Cytochrome c (Cyt c) and PARC content, concurrently. We assume that rising in PARC level may result in Cyt c degradation, and consequently suppressing/attenuating intrinsic apoptosis pathway. Besides, increasing of Casp-9 activity can be related to αS aggregates and subsequent degradation of Cyt c. To understand the mechanisms behind this using theoretical model, molecular dynamic simulation was also applied to investigate the possible interaction of Casp-9 with α-synuclein aggregates. The results showed that the interaction between Casp-9 with αS aggregates could activate Casp-9 by changing the conformation of some crucial residues.
Asunto(s)
Apoptosis , Citocromos c/metabolismo , alfa-Sinucleína/metabolismo , Sitios de Unión , Caspasa 9/química , Caspasa 9/metabolismo , Células HEK293 , Humanos , Simulación de Dinámica Molecular , Mutación Missense , Unión Proteica , Proteolisis , Transferasas/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMEN
Synthetic supramolecular polymers are used as dynamic nanoscaffolds for the activation of the apoptotic signalling enzyme caspase-9. Recruitment of caspase-9 to the nanoscaffold results in an increase in enzymatic activity due to enhanced proximity, with a bell-shaped response as a function of nanoscaffold concentration. The modularity of the system allows for dynamic regulation of enzyme activity through variation of the recruitment-motif density along the supramolecular polymer.
Asunto(s)
Caspasa 9/química , Polímeros/química , ADN/química , Nanoestructuras/química , Piridinas/químicaRESUMEN
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - RNA-guided Cas9 endonuclease system has provided a fast and efficient method for precise genome editing in diverse mammalian species, including humans. The CRISPR/Cas9 technology allows generation of modifications into site-specific locations of the selected genes in one major step by carrying deletions, insertions or DNA donor-directed precise sequence modifications. Cas9 forms a nucleoprotein complex with a sequence-specific guide RNA to create double-stranded breaks in complementary DNA target. Further, double-stranded break repair machinery leads to the intended gene modifications. The CRISPR/Cas9 system is widely used technique for genome modification, editing and other biotechnology applications, such as functional annotation, a system for visualization of specific genomic loci and transcriptional control of genes. CRISPR/Cas9-mediated manipulation of the laboratory animal genomes has contributed to the understanding of gene functions and has become a popular approach for modeling human disorders. Furthermore, the growing application of CRISPR-Cas9 system to human genes emerges as an extremely powerful technology for the molecular characterization and treatment of human disease. In this review we present the essential principles of CRISPR/Cas9 technology and the recent advances in its use in translational biomedicine.
Asunto(s)
Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Regulación de la Expresión Génica/genética , Terapia Genética/métodos , Animales , Sistemas CRISPR-Cas/fisiología , Caspasa 9/química , Caspasa 9/genética , Caspasa 9/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/genética , Modelos Animales de Enfermedad , Ingeniería Genética/métodos , Recombinación Homóloga/genética , Humanos , ARN Guía de Kinetoplastida/genéticaRESUMEN
Caspases are cysteine proteases that perform a wide variety of roles in lethal intracellular signaling and cell-death regulation. Caspase-9, the primary initiator caspase of the intrinsic apoptotic pathway, is produced as a scarcely active zymogen (Procaspase-9). Here, we describe, for the first time, at the atomistic level, conformational changes which might be correlated to the activation of Procaspase-9. Molecular dynamics simulations performed at two temperatures (310 and 410 K) provide insights about the conformational space and the time-course evolution of the geometrical and structural characteristics of Procaspase-9. At both temperatures studied, the extremal globular domains of the protein approach each other, contracting the disordered region. In both temperatures, the compact conformations hide more than 40 nm2 (about 20% of the total solvent-accessible surface area), and their radius of gyration are reduced by about 40% from the original values. At each temperature, the pathway of contraction is different, as well as the compact structures reached. In consequence, the network of stabilizing interactions at the final conformations is dissimilar. Both final conformations were evaluated in their structural compatibility with the activation models described so far. In this work, we describe mechanistically how and why the activation of Procaspase-9 is favored by apoptosome recruitment via the Caspase Activation Recruitment Domain (CARD), as it has been proposed recently by in vitro experiments.
Asunto(s)
Caspasa 9/química , Simulación de Dinámica Molecular , Activación Enzimática , HumanosRESUMEN
The aim of this study is to elucidate the detailed mechanism of endoplasmic reticulum (ER) stress-induced auditory cell death based on the function of the initiator caspases and molecular complex of necroptosis. Here, we demonstrated that ER stress initiates not only caspase-9-dependent intrinsic apoptosis along with caspase-3, but also receptor-interacting serine/threonine kinase (RIPK)1-dependent necroptosis in auditory cells. We observed the ultrastructural characteristics of both apoptosis and necroptosis in tunicamycin-treated cells under transmission electron microscopy (TEM). We demonstrated that ER stress-induced necroptosis was dependent on the induction of RIPK1, negatively regulated by caspase-8 in auditory cells. Our data suggested that ER stress-induced intrinsic apoptosis depends on the induction of caspase-9 along with caspase-3 in auditory cells. The results of this study reveal that necroptosis could exist for the alternative backup cell death route of apoptosis in auditory cells under ER stress. Interestingly, our data results in a surge in the recognition that therapies aimed at the inner ear protection effect by caspase inhibitors like zVAD-fmk might arrest apoptosis but can also have the unanticipated effect of promoting necroptosis. Thus, RIPK1-dependent necroptosis would be a new therapeutic target for the treatment of sensorineural hearing loss due to ER stress.
Asunto(s)
Apoptosis , Caspasa 8/metabolismo , Estrés del Retículo Endoplásmico , Necroptosis , Clorometilcetonas de Aminoácidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Caspasa 8/química , Caspasa 8/genética , Caspasa 9/química , Caspasa 9/genética , Caspasa 9/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Células Ciliadas Auditivas/citología , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/ultraestructura , Ratones , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Tunicamicina/farmacologíaRESUMEN
In contrast to ab-initio protein modeling methodologies, comparative modeling is considered as the most popular and reliable algorithm to model protein structure. However, the selection of the best set of templates is still a major challenge. An effective template-ranking algorithm is developed to efficiently select only the reliable hits for predicting the protein structures. The algorithm employs the pairwise as well as multiple sequence alignments of template hits to rank and select the best possible set of templates. It captures several key sequences and structural information of template hits and converts into scores to effectively rank them. This selected set of templates is used to model a target. Modeling accuracy of the algorithm is tested and evaluated on TBM-HA domain containing CASP8, CASP9 and CASP10 targets. On an average, this template ranking and selection algorithm improves GDT-TS, GDT-HA and TM_Score by 3.531, 4.814 and 0.022, respectively. Further, it has been shown that the inclusion of structurally similar templates with ample conformational diversity is crucial for the modeling algorithm to maximally as well as reliably span the target sequence and construct its near-native model. The optimal model sampling also holds the key to predict the best possible target structure.
Asunto(s)
Algoritmos , Modelos Moleculares , Dominios Proteicos , Caspasa 10/química , Caspasa 8/química , Caspasa 9/química , Biología Computacional/métodos , Conformación ProteicaRESUMEN
We discuss the relationship between the problem of protein tertiary structure prediction from the amino acid sequence and the uncertainty analysis. The algorithm presented in this paper belongs to the category of decoy-based modeling, where different known protein models are used to establish a low dimensional space via principal component analysis. The low dimensional space is utilized to perform an energy optimization via a family of very explorative particle swarm optimizers to find the global minimum. The aim of this procedure is to get a representative sample of the nonlinear equivalent region, that is, protein models that have their energy lower than a certain energy bound. The posterior analysis of this family provides very valuable information about the backbone structure of the native conformation and its possible alternate states. This methodology has the advantage of being simple and fast and can help refine the tertiary protein structure. We comprehensively illustrate the performance of our algorithm on one protein from the CASP-9 protein structure prediction experiment. We also provide a theoretical analysis of the energy landscape found in the tertiary structure protein inverse problem, explaining why model reduction techniques (principal component analysis in this case) serve to alleviate the ill-posed character of this high dimensional optimization problem. In addition, we expand the computational benchmark with a summary of other CASP-9 proteins in the Appendix.
Asunto(s)
Caspasa 9/química , Biología Computacional/métodos , Algoritmos , Secuencia de Aminoácidos , Simulación por Computador , Modelos Moleculares , Análisis de Componente Principal , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas/química , IncertidumbreRESUMEN
Apical caspases initiate and effector caspases execute apoptosis. Reagents that can distinguish between caspases, particularly apical caspases-8, 9, and 10 are scarce and generally nonspecific. Based upon a previously described large-scale screen of peptide-based caspase substrates termed HyCoSuL, we sought to develop reagents to distinguish between apical caspases in order to reveal their function in apoptotic cell death paradigms. To this end, we selected tetrapeptide-based sequences that deliver optimal substrate selectivity and converted them to inhibitors equipped with a detectable tag (activity-based probes-ABPs). We demonstrate a strong relationship between substrate kinetics and ABP kinetics. To evaluate the utility of selective substrates and ABPs, we examined distinct apoptosis pathways in Jurkat T lymphocyte and MDA-MB-231 breast cancer lines triggered to undergo cell death via extrinsic or intrinsic apoptosis. We report the first highly selective substrate appropriate for quantitation of caspase-8 activity during apoptosis. Converting substrates to ABPs promoted loss-of-activity and selectivity, thus we could not define a single ABP capable of detecting individual apical caspases in complex mixtures. To overcome this, we developed a panel strategy utilizing several caspase-selective ABPs to interrogate apoptosis, revealing the first chemistry-based approach to uncover the participation of caspase-8, but not caspase-9 or -10 in TRAIL-induced extrinsic apoptosis. We propose that using select panels of ABPs can provide information regarding caspase-8 apoptotic signaling more faithfully than can single, generally nonspecific reagents.
Asunto(s)
Caspasa 10/aislamiento & purificación , Caspasa 8/aislamiento & purificación , Caspasa 9/aislamiento & purificación , Péptidos/química , Apoptosis/genética , Caspasa 10/química , Caspasa 10/genética , Caspasa 3/química , Caspasa 3/genética , Caspasa 3/aislamiento & purificación , Caspasa 8/química , Caspasa 8/genética , Caspasa 9/química , Caspasa 9/genética , Inhibidores de Caspasas/química , Inhibidores de Caspasas/farmacología , Humanos , Células Jurkat , Cinética , Transducción de Señal , Especificidad por SustratoRESUMEN
INTRODUCTION: Scutellaria baicalensis is commonly used in Asia as an herbal medicine to treat a variety of ailments, including cancer. Wogonoside, one major constituent of S. baicalensis, can be primarily converted to wogonin through deglycosylation via enteric microbiome metabolism. MATERIALS AND METHODS: The antiproliferative effects of the glycoside (wogonoside) and its deglycosylated compound (wogonin) on a panel of human cancer cell lines from the most common solid tumors were evaluated using the MTS colorimetric assay. Cell cycle and apoptosis were determined using flow cytometry. Enzymatic activities of caspases were measured, and the interactions of wogonin and caspases were explored by a docking analysis. RESULTS: Wogonoside did not have obvious antiproliferative effects on the cancer cells. In contrast, wogonin showed significant antiproliferative activities on all the tested cancer cells. Wogonin arrested the cells in the G1 phase and significantly induced cell apoptosis. The compound also activated the expression of caspases 3 and 9. The docking results suggest that the compound forms hydrogen bonds with Phe250 and Ser251, and π-π interactions with Phe256 in caspase 3, and with Asp228 in caspase 9. CONCLUSIONS: After wogonoside deglycosylation, wogonin significantly enhanced its anticancer potential as a potent anticancer compound derived from S. baicalensis.
Asunto(s)
Flavanonas/química , Flavanonas/farmacología , Glucósidos/química , Neoplasias/tratamiento farmacológico , Extractos Vegetales/farmacología , Apoptosis/efectos de los fármacos , Caspasa 3/química , Caspasa 3/genética , Caspasa 9/química , Caspasa 9/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Glucósidos/farmacología , Glicosilación/efectos de los fármacos , Humanos , Enlace de Hidrógeno/efectos de los fármacos , Células MCF-7 , Microbiota/efectos de los fármacos , Simulación del Acoplamiento Molecular , Neoplasias/patología , Fitoterapia , Extractos Vegetales/química , Scutellaria baicalensisRESUMEN
Engineered T cell therapies show considerable promise in the treatment of refractory malignancies. Given the ability of engineered T cells to engraft and persist for prolonged periods along with unpredicted toxicities, incorporation of a suicide gene to allow selective depletion after administration is desirable. Rapamycin is a safe and widely available immunosuppressive pharmaceutical that acts by heterodimerization of FKBP12 with the FRB fragment of mTOR. The apical caspase caspase 9 is activated by homodimerization through its CARD domain. We developed a rapamycin-induced caspase 9 suicide gene. First, we showed that caspase 9 could be activated by a two-protein format with replacement of the CARD domain with both FRB and FKBP12. We next identified an optimal compact single-protein rapamycin caspase 9 (rapaCasp9) by fusing both FRB and FKBP12 with the catalytic domain of caspase 9. Functionality of rapaCasp9 when co-expressed with a CD19 CAR was demonstrated in vitro and in vivo.
Asunto(s)
Caspasa 9/genética , Regulación de la Expresión Génica/efectos de los fármacos , Expresión Génica , Genes Transgénicos Suicidas , Sirolimus/farmacología , Animales , Biomarcadores , Caspasa 9/química , Caspasa 9/metabolismo , Células Cultivadas , Citotoxicidad Inmunológica , Vectores Genéticos/genética , Humanos , Inmunofenotipificación , Ratones , Dominios y Motivos de Interacción de Proteínas , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Caspase-9 is a critical factor in the initiation of apoptosis and as a result is tightly regulated by many mechanisms. Caspase-9 contains a Caspase Activation and Recruitment Domain (CARD), which enables caspase-9 to form a tight interaction with the apoptosome, a heptameric activating platform. The caspase-9 CARD has been thought to be principally involved in recruitment to the apoptosome, but its roles outside this interaction have yet to be uncovered. In this work, we show that the CARD is involved in physical interactions with the catalytic core of caspase-9 in the absence of the apoptosome; this interaction requires a properly formed caspase-9 active site. The active sites of caspases are composed of four extremely mobile loops. When the active-site loops are not properly ordered, the CARD and core domains of caspase-9 do not interact and behave independently, like loosely tethered beads. When the active-site loop bundle is properly ordered, the CARD domain interacts with the catalytic core, forming a single folding unit. Taken together, these findings provide mechanistic insights into a new level of caspase-9 regulation, prompting speculation that the CARD may also play a role in the recruitment or recognition of substrate.
Asunto(s)
Caspasa 9/química , Caspasa 9/metabolismo , Dominio de Reclutamiento y Activación de Caspasas/fisiología , Pliegue de Proteína , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Caspasa 9/genética , Dominio Catalítico/genética , Activación Enzimática/genética , Humanos , Modelos Moleculares , Mutación Missense , Unión Proteica , Multimerización de Proteína/genéticaRESUMEN
Macroautophagy/autophagy has emerged as a resistance mechanism to anticancer drug treatments that induce metabolic stress. Certain tumors, including a subset of KRAS-mutant NSCLCs have been shown to be addicted to autophagy, and potentially vulnerable to autophagy inhibition. Currently, autophagy inhibition is being tested in the clinic as a therapeutic component for tumors that utilize this degradation process as a drug resistance mechanism. The current study provides evidence that HSP90 (heat shock protein 90) inhibition diminishes the expression of ATG7, thereby impeding the cellular capability of mounting an effective autophagic response in NSCLC cells. Additionally, an elevation in the expression level of CASP9 (caspase 9) prodomain in KRAS-mutant NSCLC cells surviving HSP90 inhibition appears to serve as a cell survival mechanism. Initial characterization of this survival mechanism suggests that the altered expression of CASP9 is mainly ATG7 independent; it does not involve the apoptotic activity of CASP9; and it localizes to a late endosomal and pre-lysosomal phase of the degradation cascade. HSP90 inhibitors are identified here as a pharmacological approach for targeting autophagy via destabilization of ATG7, while an induced expression of CASP9, but not its apoptotic activity, is identified as a resistance mechanism to the cellular stress brought about by HSP90 inhibition.
Asunto(s)
Autofagia , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Caspasa 9/metabolismo , Resistencia a Antineoplásicos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Neoplasias Pulmonares/metabolismo , Autofagia/efectos de los fármacos , Proteína 7 Relacionada con la Autofagia/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Caspasa 9/química , Línea Celular Tumoral , Citoprotección/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Estabilidad de Enzimas/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Neoplasias Pulmonares/patología , Mutación/genética , Dominios Proteicos , Triazoles/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
L-mimosine is a rare plant amino acid extracted from Mimosa or Leucaena spp., and it has been reported to exhibit antitumor activity in a number of types of cancer. However, the underlying mechanisms remain to be clarified. In the present study, the effect of Lmimosine was investigated in human osteosarcoma cells. A Cell Counting Kit8 assay and flow cytometry were used for toxicity detection. Hoechst staining and transmission electron microscopy (TEM), in addition to western blot analysis, were used for the examination of the associated mechanisms. The results of the present study indicated that Lmimosine significantly inhibited cell proliferation by inducing cellular apoptosis in osteosarcoma cells. The Hoechst staining results and TEM revealed that nuclear damage increased with the concentration increase in Lmimosine, as did the formation of apoptotic bodies. Additionally, the results of the western blot analysis confirmed that the treatment of cells with Lmimosine was accompanied by increasing expression of cleaved caspase9. Lmimosineinduced apoptosis was inhibited by the caspase9 inhibitor ZLEHDFMK. In addition, the extracellular signalregulated kinase (ERK) signaling pathway was suppressed following treatment with Lmimosine. In conclusion, the results of the present study suggested that Lmimosine induced apoptosis via the mitochondrial apoptotic pathway. The ERK signaling pathway was indicated to be an additional mechanism underlying apoptosis induction. The results provided evidence for the use of Lmimosine as a promising candidate for osteosarcoma therapy.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasa 9/metabolismo , Mimosina/farmacología , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Caspasa 9/química , Inhibidores de Caspasas/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Microscopía Electrónica de Transmisión , Osteosarcoma/metabolismo , Osteosarcoma/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Caspases, the cysteine proteases which facilitate the faithful execution of apoptosis, are tightly regulated by a number of mechanisms including phosphorylation. In response to cAMP, PKA phosphorylates caspase-9 at three sites preventing caspase-9 activation, and suppressing apoptosis progression. Phosphorylation of caspase-9 by PKA at the functionally relevant site Ser-183 acts as an upstream block of the apoptotic cascade, directly inactivating caspase-9 by a two-stage mechanism. First, Ser-183 phosphorylation prevents caspase-9 self-processing and directly blocks substrate binding. In addition, Ser-183 phosphorylation breaks the fundamental interactions within the caspase-9 core, promoting disassembly of the large and small subunits. This occurs despite Ser-183 being a surface residue distal from the interface between the large and small subunits. This phosphorylation-induced disassembly promotes the formation of ordered aggregates around 20 nm in diameter. Similar aggregates of caspase-9 have not been previously reported. This two-stage regulatory mechanism for caspase-9 has likewise not been reported previously but may be conserved across the caspases.
Asunto(s)
Caspasa 3/química , Caspasa 9/química , Proteínas Quinasas Dependientes de AMP Cíclico/química , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 9/genética , Caspasa 9/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , FosforilaciónRESUMEN
Diterpenes are compounds with complex structure and due to their unique carbon skeleton and interesting biological activities, have been the focus of continuous studies for the development of new anticancer agents. The plants of the genus Calceolaria (Scrophulariaceae family), native of South America have also yielded several new diterpenes with the scopadulane skeleton, such as thyrsiflorin A. The present study was undertaken to investigate the effect of the semisynthetic compound, demalonyl thyrsiflorin A on human melanoma cells. In A375 cells compound demalonyl thyrsiflorin A showed a clear dose-response relationship in the range of 6.25-50µM concentrations. In addition, we demonstrated an apoptotic response after treatment of cancer cells with this semisynthetic phenolic labdane diterpene at 6.25 and 12.5µM concentrations that probably involves the reduction of Hsp70 expression and reactive oxygen species production. Alternatively, the inhibition of the caspase cascade at higher concentrations, 25 and 50µM, correlated with additional reactive oxygen species increase, probably switched the mode of demalonyl thyrsiflorin A-induced cell death from apoptosis to necrosis.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Diterpenos/farmacología , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Melanoma/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/agonistas , Caspasa 3/química , Caspasa 3/metabolismo , Caspasa 9/química , Caspasa 9/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayo Cometa , Fragmentación del ADN/efectos de los fármacos , Diseño de Fármacos , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Etiquetado Corte-Fin in Situ , Melanoma/metabolismo , Melanoma/patología , Necrosis , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Concentración Osmolar , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Apoptotic signaling pathways are altered in numerous pathologies such as cancer. In this scenario, caspase-9/PP2Acα interaction constitutes a key target with pharmacological interest to re-establish apoptosis in tumor cells. Very recently, a short peptide (C9h) known to disrupt caspase-9/PP2Acα interaction with subsequent apoptosis induction was described. Here, we prepared two sets of mesoporous silica nanoparticles loaded with safraninâ O (S2) or with C9h peptide (S4) and functionalized with ϵ-polylysine as capping unit. Aqueous suspensions of both nanoparticles showed negligible cargo release whereas in the presence of pronase, a marked delivery of safraninâ O or C9h was observed. Confocal microscopy studies carried out with HeLa cells indicated that both materials were internalized and were able to release their entrapped cargos. Besides, a marked decrease in HeLa cell viability (ca. 50 %) was observed when treated with C9h-loaded S4 nanoparticles. Moreover, S4 provides peptide protection from degradation additionally allowing for a dose reduction to observe an apoptotic effect when compared with C9h alone or in combination with a cell-penetrating peptide (i.e., Mut3DPT-C9h). Flow cytometry studies, by means of Annexin V-FITC staining, showed the activation of apoptotic pathways in HeLa as a consequence of S4 internalization, release of C9h peptide and disruption of caspase-9/PP2Acα interaction.
Asunto(s)
Nanopartículas/química , Péptidos/química , Polilisina/química , Dióxido de Silicio/química , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , Caspasa 9/química , Caspasa 9/metabolismo , Dicroismo Circular , Portadores de Fármacos/síntesis química , Portadores de Fármacos/química , Células HeLa , Humanos , Microscopía Confocal , Péptidos/toxicidad , Fenazinas/química , Fenazinas/toxicidad , Porosidad , Proteína Fosfatasa 2/química , Proteína Fosfatasa 2/metabolismoRESUMEN
BACKGROUND: Isofraxidin (IF), a natural coumarin compound, has been reported to possess anti-cancer activity in human liver cancer. However, whether IF is involved in the regulation of colorectal cancer tumorigenesis and development has been not well elucidated. METHODS: The cell proliferation were assessed by Cell Counting Kit-8 (CCK-8) and colony formation test, respectively. The transwell assays were conducted to estimate cell migration and invasion abilities. Further, cell apoptosis was evaluated by confocal microscopy analysis, flow cytometry detection and TdT-mediated dUTP Nick-End Labeling (TUNEL) method. Western blot were performed to detect the expression of related protein. RESULTS: Herein, the result indicated that IF remarkably bated cell proliferation in human colorectal cancer cells HT-29 and SW-480 in a dose- and time-dependent manner. In addition, IF treatment showed obvious inhibitory activity to cell colony formation in HT-29 and SW-480 cells. Confocal microscopy analysis and flow cytometry detection revealed that IF dramatically induced cell apoptosis in HT-29 and SW-480 cells compared with the control. And IF markedly decreased the expression of anti-apoptotic protein bcl-2, whereas the expression of pro-apoptotic proteins, including caspase-3, caspase-9 and bax, notably increased in HT-29 and SW-480 cells. Besides, IF blocked Akt pathway via inhibition expression of p-Akt. Furthermore, MK2206, an Akt inhibitor, could inhibit cell colony formation and induced apoptosis. This effect is even more obvious in the presence of MK2206 and IF compared to that of either agent alone. CONCLUSIONS: Together, the present study reports a novel use of IF in mitigating human colorectal cancer proliferation and inducing apoptosis via blockage of Akt pathway.