RESUMEN
The endogenous protease furin is a key protein in many different diseases, such as cancer and infections. For this reason, a wide range of studies has focused on targeting furin from a therapeutic point of view. Our main objective consisted of identifying new compounds that could enlarge the furin inhibitor arsenal; secondarily, we assayed their adjuvant effect in combination with a known furin inhibitor, CMK, which avoids the SARS-CoV-2 S protein cleavage by means of that inhibition. Virtual screening was carried out to identify potential furin inhibitors. The inhibition of physiological and purified recombinant furin by screening selected compounds, Clexane, and these drugs in combination with CMK was assayed in fluorogenic tests by using a specific furin substrate. The effects of the selected inhibitors from virtual screening on cell viability (293T HEK cell line) were assayed by means of flow cytometry. Through virtual screening, Zeaxanthin and Kukoamine A were selected as the main potential furin inhibitors. In fluorogenic assays, these two compounds and Clexane inhibited both physiological and recombinant furin in a dose-dependent way. In addition, these compounds increased physiological furin inhibition by CMK, showing an adjuvant effect. In conclusion, we identified Kukoamine A, Zeaxanthin, and Clexane as new furin inhibitors. In addition, these drugs were able to increase furin inhibition by CMK, so they could also increase its efficiency when avoiding S protein proteolysis, which is essential for SARS-CoV-2 cell infection.
Asunto(s)
Clorometilcetonas de Aminoácidos/farmacología , Enoxaparina/farmacología , Furina/antagonistas & inhibidores , Espermina/análogos & derivados , Zeaxantinas/farmacología , Clorometilcetonas de Aminoácidos/química , Clorometilcetonas de Aminoácidos/metabolismo , COVID-19/transmisión , COVID-19/virología , Dominio Catalítico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Enoxaparina/química , Enoxaparina/metabolismo , Furina/química , Furina/metabolismo , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/farmacología , Proteolisis , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiología , Espermina/química , Espermina/metabolismo , Espermina/farmacología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus , Replicación Viral , Zeaxantinas/química , Zeaxantinas/metabolismoRESUMEN
A pre-existing, allosteric equilibrium between closed (E*) and open (E) conformations of the active site influences the level of activity in the trypsin fold and defines ligand binding according to the mechanism of conformational selection. Using the clotting protease thrombin as a model system, we investigate the molecular determinants of the E*-E equilibrium through rapid kinetics and X-ray structural biology. The equilibrium is controlled by three residues positioned around the active site. W215 on the 215-217 segment defining the west wall of the active site controls the rate of transition from E to E* through hydrophobic interaction with F227. E192 on the opposite 190-193 segment defining the east wall of the active site controls the rate of transition from E* to E through electrostatic repulsion of E217. The side chain of E217 acts as a lever that moves the entire 215-217 segment in the E*-E equilibrium. Removal of this side chain converts binding to the active site to a simple lock-and-key mechanism and freezes the conformation in a state intermediate between E* and E. These findings reveal a simple framework to understand the molecular basis of a key allosteric property of the trypsin fold.
Asunto(s)
Aminoácidos/metabolismo , Trombina/química , Trombina/metabolismo , Regulación Alostérica , Clorometilcetonas de Aminoácidos/metabolismo , Humanos , Cinética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Unión Proteica , Conformación ProteicaRESUMEN
We previously developed a test for detecting naturally occurring protein-induced skin sensitization based on the markers and criteria of the human cell-line activation test (h-CLAT) and showed that the h-CLAT was useful for assessing the allergenic potency of proteins. However, test proteins were contaminated with varying amounts of lipopolysaccharide (LPS), which might have contributed to the stimulation of CD86 and CD54 expression. In this study, we developed a method to exclude the effects of LPS in the assessment of skin sensitization by naturally occurring proteins. We tested two inhibitors [the caspase-1 inhibitor acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-cmk; hereafter referred to as YVAD), which can mitigate the LPS-induced increases in CD54 expression, and polymyxin B (PMB), which suppresses the effect of LPS by binding to its lipid moiety (i.e., the toxic component of LPS)]. After a 24 hr exposure, YVAD and PMB reduced LPS-induced CD86 and CD54 expression. In particular, the effect of PMB was dependent upon pre-incubation time and temperature, with the most potent effect observed following pre-incubation at 37°C for 24 hr. Moreover, only pre-incubation with cell-culture medium (CCM) at 37°C for 24 hr showed an inhibitory effect similar to that of PMB, with this result possibly caused by components of CCM binding to LPS. Similar effects were observed in the presence of ovalbumin (with 1070 EU/mg LPS) and ovomucoid, and lysozyme (with 2.82 and 0.234 EU/mg LPS, respectively) in CCM. These results indicated that PMB and CCM effectively eliminated the effects of LPS during assessment of protein allergenicity, thereby allowing a more accurate evaluation of the potential of proteins to induce skin sensitization.
Asunto(s)
Clorometilcetonas de Aminoácidos/farmacología , Hipersensibilidad/etiología , Hipersensibilidad/inmunología , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/inmunología , Polimixina B/farmacología , Proteínas/efectos adversos , Proteínas/inmunología , Pruebas Cutáneas/métodos , Piel/inmunología , Clorometilcetonas de Aminoácidos/administración & dosificación , Clorometilcetonas de Aminoácidos/metabolismo , Antígeno B7-2/genética , Antígeno B7-2/inmunología , Antígeno B7-2/metabolismo , Medios de Cultivo , Expresión Génica/efectos de los fármacos , Humanos , Inmunización , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/inmunología , Molécula 1 de Adhesión Intercelular/metabolismo , Lipopolisacáridos/efectos adversos , Lipopolisacáridos/metabolismo , Muramidasa , Ovalbúmina , Ovomucina , Polimixina B/administración & dosificación , Polimixina B/metabolismo , Unión Proteica , Células THP-1 , Temperatura , Factores de TiempoRESUMEN
Human α-thrombin is a serine protease with dual functions. Thrombin acts as a procoagulant, cleaving fibrinogen to make the fibrin clot, but when bound to thrombomodulin (TM), it acts as an anticoagulant, cleaving protein C. A minimal TM fragment consisting of the fourth, fifth, and most of the sixth EGF-like domain (TM456m) that has been prepared has much improved solubility, thrombin binding capacity, and anticoagulant activity versus those of previous TM456 constructs. In this work, we compare backbone amide exchange of human α-thrombin in three states: apo, D-Phe-Pro-Arg-chloromethylketone (PPACK)-bound, and TM456m-bound. Beyond causing a decreased level of amide exchange at their binding sites, TM and PPACK both cause a decreased level of amide exchange in other regions including the γ-loop and the adjacent N-terminus of the heavy chain. The decreased level of amide exchange in the N-terminus of the heavy chain is consistent with the historic model of activation of serine proteases, which involves insertion of this region into the ß-barrel promoting the correct conformation of the catalytic residues. Contrary to crystal structures of thrombin, hydrogen-deuterium exchange mass spectrometry results suggest that the conformation of apo-thrombin does not yet have the N-terminus of the heavy chain properly inserted for optimal catalytic activity, and that binding of TM allosterically promotes the catalytically active conformation.
Asunto(s)
Trombina/química , Trombina/metabolismo , Trombomodulina/química , Trombomodulina/metabolismo , Clorometilcetonas de Aminoácidos/química , Clorometilcetonas de Aminoácidos/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Medición de Intercambio de Deuterio , Humanos , Espectrometría de Masas , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trombina/genética , Trombomodulina/genéticaRESUMEN
Protease inhibitors affecting the activity of the proteasome were reported to induce programmed cell death (apoptosis) in some mammalian cell lines. Proteasome activity can be suppressed by specific peptide derivatives and by N-tosyl-lysine-chloromethyl-ketone (TLCK) and N-tosyl-phenylalanine-chloromethyl-ketone (TPCK), which affect the trypsine- and chymotrypsine-like activities of the proteasome, respectively. Particularly TLCK and TPCK caused necrotic cell death in the unicellular green alga Chlamydomonas reinhardtii. As a control, the effects of these protease inhibitors on the survival of human WISH cells were also studied. Bleaching of the Chlamydomonas cells after addition of TLCK or TPCK indicated that reactive oxygen species (ROS) were involved in this process. Indeed, increased levels of ROS were detected in Chlamydomonas cells treated with TLCK or TPCK. Furthermore, cell death induced by these protease inhibitors was accelerated by illumination and prevented or slowed down by scavengers of ROS.
Asunto(s)
Apoptosis/efectos de los fármacos , Chlamydomonas reinhardtii/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Clorometilcetonas de Aminoácidos/metabolismo , Amnios/citología , Línea Celular , Chlamydomonas reinhardtii/citología , Chlamydomonas reinhardtii/metabolismo , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
Local anesthetics have been reported to induce apoptosis in various cell lines. In this study, we showed that bupivacaine also induced apoptosis in DTK-SME cells, a vimentin(+)/AE1(+)/CK7(+)/HSP27(+), tumorigenic, immortalized, canine mammary tumor cell line. Bupivacaine induced apoptosis in DTK-SME cells in a time- and concentration-dependent manner. Apoptosis-associated morphological changes, including cell shrinkage and rounding, chromatin condensation, and formation of apoptotic bodies, were observed in the bupivacaine-treated DTK-SME cells. Apoptosis was further confirmed with annexin V staining, TUNEL staining, and DNA laddering assays. At the molecular level, the activation of caspases-3, -8, and -9 corresponded well to the degree of DNA fragmentation triggered by bupivacaine. We also demonstrated that the pan-caspase inhibitor, z-VAD-fmk, only partially inhibited the apoptosis induced by bupivacaine. Moreover, treated cells increased expression of endonuclease G, a death effector that acts independently of caspases. Our data suggested that bupivacaine-induced apoptosis occurs through both caspase-dependent and caspase-independent apoptotic pathways.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Bupivacaína/farmacología , Caspasas/metabolismo , Enfermedades de los Perros/tratamiento farmacológico , Neoplasias Mamarias Animales/tratamiento farmacológico , Clorometilcetonas de Aminoácidos/metabolismo , Animales , Inhibidores de Caspasas/metabolismo , Línea Celular Tumoral , Fragmentación del ADN/efectos de los fármacos , Enfermedades de los Perros/etiología , Perros , Femenino , Neoplasias Mamarias Animales/etiologíaRESUMEN
Dithiocarbamates (DTCs) exhibit a broad spectrum of antitumor activities, however, their molecular mechanisms of antitumor have not yet been elucidated. Previously, we have synthesized a series of novel dithiocarbamate derivatives. These DTCs were examined for cytotoxic activities against five human cancer cell lines. In this study, one of dithiocarbamate (DTC1) with higher potential for HeLa cells was chosen to investigate molecular mechanisms for its anti-tumor activities. DTC1 could inhibit proliferation, and highly induce apoptosis in HeLa cells by activating caspase-3, -6 and -9; moreover, activities of caspase-3, -6 and -9 were inhibited by pan-caspase inhibitor, Z-VAD-FMK. Furthermore, DTC1 decreased the levels of Bcl-2 and Bcl-xL, and increased expression of cytosol cytochrome c, Bak, Bax and p53 in a time-dependent manner but had no effect on the level of Rb. It was shown that DTC1 induced HeLa cells apoptosis through a p53-dependent pathway as tested by the wild type p53 inhibitor, pifithrin-α. Additionally, the relative expression of E6 and E7 were evaluated in HPV18-positive (HeLa cells) by real-time PCR and western blotting. The results firstly demonstrated that DTC1 suppressed both expression of E6 mRNA and E6 oncoprotein, but had no effect on the expression of E7 mRNA and protein in HPV18. Our results suggested that DTC1 may serve as novel chemotherapeutic agents in the treatment of cervical cancer and potential anti-HPV virus candidates that merit further studies.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Etilenobis(ditiocarbamatos)/farmacología , Proteínas Oncogénicas Virales/metabolismo , Clorometilcetonas de Aminoácidos/metabolismo , Antineoplásicos/química , Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasa 3/metabolismo , Caspasa 6/metabolismo , Caspasa 9/metabolismo , Inhibidores de Caspasas/metabolismo , Proliferación Celular/efectos de los fármacos , Citocromos c/metabolismo , Etilenobis(ditiocarbamatos)/química , Células HeLa , Humanos , Proteínas E7 de Papillomavirus/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Anion transporters based on small molecules have received attention as therapeutic agents because of their potential to disrupt cellular ion homeostasis. However, a direct correlation between a change in cellular chloride anion concentration and cytotoxicity has not been established for synthetic ion carriers. Here we show that two pyridine diamide-strapped calix[4]pyrroles induce coupled chloride anion and sodium cation transport in both liposomal models and cells, and promote cell death by increasing intracellular chloride and sodium ion concentrations. Removing either ion from the extracellular media or blocking natural sodium channels with amiloride prevents this effect. Cell experiments show that the ion transporters induce the sodium chloride influx, which leads to an increased concentration of reactive oxygen species, release of cytochrome c from the mitochondria and apoptosis via caspase activation. However, they do not activate the caspase-independent apoptotic pathway associated with the apoptosis-inducing factor. Ion transporters, therefore, represent an attractive approach for regulating cellular processes that are normally controlled tightly by homeostasis.
Asunto(s)
Proteínas de Transporte de Anión/metabolismo , Apoptosis/efectos de los fármacos , Cloruros/farmacología , Bibliotecas de Moléculas Pequeñas/metabolismo , Clorometilcetonas de Aminoácidos/química , Clorometilcetonas de Aminoácidos/metabolismo , Animales , Proteínas de Transporte de Anión/química , Calixarenos/química , Calixarenos/metabolismo , Caspasas/química , Caspasas/metabolismo , Línea Celular , Cloruros/química , Citocromos c/metabolismo , Diamida/química , Células HCT116 , Células HeLa , Humanos , Transporte Iónico , Liposomas/química , Mitocondrias/metabolismo , Porfirinas/química , Porfirinas/metabolismo , Piridinas/química , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Chronic hepatitis C virus (HCV) infection results in progressive liver fibrosis leading to cirrhosis and liver cancer. The mechanism for this remains unclear but hepatocyte apoptosis is thought to play a major role. Hepatocyte apoptosis in human liver tissue was determined by immunohistochemistry for cytokeratin 18 (M30 CytoDEATH) and cleaved poly(ADP-ribose) polymerase (PARP). In vitro studies were performed with replication-defective recombinant adenoviruses expressing HCV proteins (rAdHCV) to study the effects of HCV on cell death in Huh7 cells, primary mouse hepatocytes (PMoHs) and primary human hepatocytes (PHHs). Cell viability and apoptosis were studied using crystal violet assays and Western blots probed for cleaved caspase-3 and cleaved PARP, with and without treatment with the pan-caspase inhibitor Q-VD-OPh and necrostatin-1. Liver tissue of HCV-infected patients expressed elevated levels of apoptotic markers compared with HCV-negative patients. rAdHCV infection reduced cell viability compared with uninfected controls and cells infected with control virus (rAdGFP). Huh7, PMoHs and PHHs infected with rAdHCV showed significantly increased levels of apoptotic markers compared with uninfected controls and rAdGFP-infected cells. In rAdHCV-infected Huh7, treatment with Q-VD-OPh and necrostatin-1 both improved cell viability. Q-VD-Oph also reduced cleaved PARP in rAdHCV-infected Huh7 and PMoHs. Hepatocyte apoptosis is known to be increased in the livers of HCV-infected patients. HCV promoted cell death in primary and immortalized hepatocytes, and this was inhibited by Q-VD-OPh and necrostatin-1. These findings indicate that HCV-induced cell death occurs by both apoptosis and necroptosis, and provide new insights into the mechanisms of HCV-induced liver injury.
Asunto(s)
Apoptosis , Hepacivirus/fisiología , Hepatitis C/patología , Hepatocitos/fisiología , Hepatocitos/virología , Necrosis , Clorometilcetonas de Aminoácidos/metabolismo , Animales , Caspasas/análisis , Supervivencia Celular , Inhibidores Enzimáticos/metabolismo , Hepatocitos/efectos de los fármacos , Humanos , Imidazoles/metabolismo , Indoles/metabolismo , Ratones , Ratones Endogámicos C57BL , Quinolinas/metabolismoRESUMEN
Thrombin is the central protease in the cascade of blood coagulation proteases. The structure of thrombin consists of a double ß-barrel core surrounded by connecting loops and helices. Compared to chymotrypsin, thrombin has more extended loops that are thought to have arisen from insertions in the serine protease that evolved to impart greater specificity. Previous experiments showed thermodynamic coupling between ligand binding at the active site and distal exosites. We present a combined approach of molecular dynamics (MD), accelerated molecular dynamics (AMD), and analysis of the residual local frustration of apo-thrombin and active-site-bound (PPACK-thrombin). Community analysis of the MD ensembles identified changes upon active site occupation in groups of residues linked through correlated motions and physical contacts. AMD simulations, calibrated on measured residual dipolar couplings, reveal that upon active site ligation, correlated loop motions are quenched, but new ones connecting the active site with distal sites where allosteric regulators bind emerge. Residual local frustration analysis reveals a striking correlation between frustrated contacts and regions undergoing slow time scale dynamics. The results elucidate a motional network that probably evolved through retention of frustrated contacts to provide facile conversion between ensembles of states.
Asunto(s)
Simulación de Dinámica Molecular , Trombina/química , Regulación Alostérica , Clorometilcetonas de Aminoácidos/química , Clorometilcetonas de Aminoácidos/metabolismo , Dominio Catalítico , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Trombina/metabolismoRESUMEN
BACKGROUND: Numerous studies have demonstrated that autophagy plays a vital role in maintaining cellular homeostasis. Interestingly, several anticancer agents were found to exert their anticancer effects by triggering autophagy. Emerging data suggest that autophagy represents a novel mechanism that can be exploited for therapeutic benefit. Pharmacologically active natural compounds such as those from marine, terrestrial plants and animals represent a promising resource for novel anticancer drugs. There are several prominent examples from the past proving the success of natural products and derivatives exhibiting anticancer activity. Helenalin, a sesquiterpene lactone has been demonstrated to have potent anti-inflammatory and antitumor activity. Albeit previous studies demonstrating helenalin's multi modal action on cellular proliferative and apoptosis, the mechanisms underlying its action are largely unexplained. METHODS: To deduce the mechanistic action of helenalin, cancer cells were treated with the drug at various concentrations and time intervals. Using western blot, FACS analysis, overexpression and knockdown studies, cellular signaling pathways were interrogated focusing on apoptosis and autophagy markers. RESULTS: We show here that helenalin induces sub-G1 arrest, apoptosis, caspase cleavage and increases the levels of the autophagic markers. Suppression of caspase cleavage by the pan caspase inhibitor, Z-VAD-fmk, suppressed induction of LC3-B and Atg12 and reduced autophagic cell death, indicating caspase activity was essential for autophagic cell death induced by helenalin. Additionally, helenalin suppressed NF-κB p65 expression in a dose and time dependent manner. Exogenous overexpression of p65 was accompanied by reduced levels of cell death whereas siRNA mediated suppression led to augmented levels of caspase cleavage, autophagic cell death markers and increased cell death. CONCLUSIONS: Taken together, these results show that helenalin mediated autophagic cell death entails inhibition of NF-κB p65, thus providing a promising approach for the treatment of cancers with aberrant activation of the NF-κB pathway.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Arnica/química , Autofagia/efectos de los fármacos , Neoplasias/metabolismo , Fitoterapia , Receptores Similares a Lectina de Células NK/antagonistas & inhibidores , Sesquiterpenos/farmacología , Clorometilcetonas de Aminoácidos/metabolismo , Antineoplásicos Fitogénicos/uso terapéutico , Apoptosis/efectos de los fármacos , Proteína 12 Relacionada con la Autofagia , Caspasas/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Fase G1/efectos de los fármacos , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Neoplasias/tratamiento farmacológico , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , ARN Interferente Pequeño/metabolismo , Sesquiterpenos/uso terapéutico , Sesquiterpenos de Guayano , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismoRESUMEN
Epidemiological, preclinical and clinical studies have supported the role of selenocompounds as potential cancer chemopreventive and chemotherapeutic agents. In this study, a novel selenophene-based compound, 1,4-diselenophene-1,4-diketone (DSeD), has been synthesized by Double Friedel-Crafts reaction and identified as a potent antiproliferative agent against a panel of six human caner cell lines. Despite this potency, DSeD was relatively nontoxic toward human normal cells, HS68 fibroblasts and HK-2 kidney cells. These results suggest that DSeD possesses great selectivity between cancer and normal cells. Induction of apoptosis in human melanoma A375 cells by DSeD was evidenced by accumulation of sub-G1 cell population, DNA fragmentation and nuclear condensation. Activation of caspase-9 and depletion of mitochondrial membrane potential indicated the initiation of the mitochondria-mediated apoptosis pathway. Pretreatment of cells with general caspase inhibitor z-VAD-fmk and caspase-9 inhibitor z-LEHD-fmk significantly suppressed the cell apoptosis, demonstrating the important roles of caspase and mitochondria in DSeD-induced apoptotic cell death. Furthermore, DSeD-induced apoptosis was found independent of reactive oxygen species generation. Taken together, our results suggest that DSeD induces caspase-dependent apoptosis in A375 cells through activation of mitochondria-mediated apoptosis pathway.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de Caspasas , Cetonas/química , Cetonas/farmacología , Mitocondrias/efectos de los fármacos , Compuestos de Organoselenio/química , Compuestos de Organoselenio/farmacología , Clorometilcetonas de Aminoácidos/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Caspasas/análisis , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Radicales Libres/metabolismo , Humanos , Melanoma/metabolismo , Melanoma/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Thrombin is a serine proteinase that plays a fundamental role in coagulation. In this study, we address the effects of ligand site recognition by alpha-thrombin on conformation and energetics in solution. Active site occupation induces large changes in secondary structure content in thrombin as shown by circular dichroism. Thrombin-D-Phe-Pro-Arg-chloromethyl ketone (PPACK) exhibits enhanced equilibrium and kinetic stability compared to free thrombin, whose difference is rooted in the unfolding step. Small-angle X-ray scattering (SAXS) measurements in solution reveal an overall similarity in the molecular envelope of thrombin and thrombin-PPACK, which differs from the crystal structure of thrombin. Molecular dynamics simulations performed with thrombin lead to different conformations than the one observed in the crystal structure. These data shed light on the diversity of thrombin conformers not previously observed in crystal structures with distinguished catalytic and conformational behaviors, which might have direct implications on novel strategies to design direct thrombin inhibitors.
Asunto(s)
Clorometilcetonas de Aminoácidos/química , Clorometilcetonas de Aminoácidos/metabolismo , Simulación de Dinámica Molecular , Trombina/química , Trombina/metabolismo , Sitios de Unión , Humanos , Dispersión del Ángulo Pequeño , Termodinámica , Rayos XRESUMEN
The study aimed to evaluate the effects of ß-escin on human cholangiocarcinoma cell lines (QBC939, Sk-ChA-1 and MZ-ChA-1) and to explore its mechanisms. Cell growth, cell cycle and apoptosis were investigated, respectively, by MTT assay, single PI and FITC/PI double-staining flow cytometry, and fluorescence microscopy. The protein expression was determined by western blotting. The study revealed that ß-escin inhibited cholangiocarcinoma cell growth in a dose- and time-dependent manner, and the cell cycle of QBC939 and Sk-ChA-1 cells was arrested in the G2/M phase, and MZ-ChA-1 cells in G1 phase. Apoptosis of the three cholangiocarcinoma cell lines induced by ß-escin was associated with the collapse of the mitochondrial membrane potential and the activation of caspase-3. The apoptotic effect of ß-escin was suppressed by pancaspase inhibitor z-VAD-fmk. Molecular dissection revealed that the antiapoptotic protein bcl-2 was down-regulated after cholangiocarcinoma cell lines were treated with ß-escin, while the protein levels of bax and p53 were unchanged. Apoptosis was accompanied by an increase in reactive oxygen species (ROS). These results suggest that ß-escin induces apoptosis of cholangiocarcinoma cells through an intrinsic mitochondrial caspase-dependent pathway, and the increase in the bax/bcl-2 ratio and ROS may play important roles in ß-escin-induced apoptosis of cholangiocarcinoma cells.
Asunto(s)
Aesculus/química , Apoptosis/efectos de los fármacos , Neoplasias de los Conductos Biliares/prevención & control , Conductos Biliares Intrahepáticos/efectos de los fármacos , Colangiocarcinoma/prevención & control , Escina/uso terapéutico , Fitoterapia , Clorometilcetonas de Aminoácidos/metabolismo , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Neoplasias de los Conductos Biliares/metabolismo , Conductos Biliares Intrahepáticos/metabolismo , Conductos Biliares Intrahepáticos/patología , Caspasa 3/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colangiocarcinoma/metabolismo , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Escina/farmacología , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Proteínas Nucleares/metabolismo , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Tumoral p73 , Proteínas Supresoras de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Tanshinone IIA (Tan-IIA) is extracted from Danshen, Salviae miltiorrhizae Radix, which has been widely adopted in traditional herbal medicine to treat cardiovascular and hepatic diseases. Tan-IIA induces apoptosis and inhibits proliferation in human hepatocellular carcinoma (HCC) cells. However, the molecular mechanisms of Tan-IIA on human HCC cells are not understood clearly. In the present study, the cytotoxicity of Tan-IIA as well as its molecular mechanisms in human HCC J5 cells was investigated. The cytotoxicity was assayed by MTT. The protein expression of p53, p21, Bax, Bcl-2, Cdc25c, Cdc2, calreticulin, caspase 12, GADD153, caspase 3 and beta-actin in J5 cells were determined by Western blotting. The cell cycles were analyzed by FACS. The protein expression of caspase 12, GADD1533 and caspase 3 were detected by immunocytochemical staining. The results showed that Tan-IIA inhibited J5 cells in a dose- and time-dependent manner. The protein expression of p53, p21, Bax, calreticulin, caspase 12, caspase 3 and GADD153 were increased, but Bcl-2, Cdc25c and Cdc2 were decreased in J5 cells. In addition, the results also showed that Tan-IIA arrested J5 cells in the G2/M phase. Immunocytochemistry staining showed that J5 cells treated with Tan-IIA up-regulated the protein expression of caspase 12, 3 and GADD153. Taken together, the findings suggest that Tan-IIA inhibits and induces apoptosis in J5 cells through novel molecular targets, calreticulin, caspase 12 and GADD153.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Calreticulina/metabolismo , Carcinoma Hepatocelular/tratamiento farmacológico , Caspasa 12/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Fenantrenos/farmacología , Factor de Transcripción CHOP/metabolismo , Abietanos , Clorometilcetonas de Aminoácidos/metabolismo , Animales , Antineoplásicos Fitogénicos/uso terapéutico , Apoptosis/efectos de los fármacos , Inhibidores de Caspasas , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/metabolismo , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Humanos , Masculino , Mitocondrias/metabolismo , Fenantrenos/uso terapéuticoRESUMEN
Doxorubicin (Dox) is widely used to treat a variety of tumors. However, resistance to this drug is common, making successful treatment more difficult. Previously, we introduced a novel phytosphingosine derivative, N,N-dimethyl phytosphingosine (DMPS), as a potent anticancer therapeutic agent in human leukemia cells. This study was performed to investigate whether DMPS can sensitize HL-60/MX2, a multidrug-resistant variant of HL-60, to Dox-induced apoptosis. Low concentrations of DMPS sensitized HL-60/MX2 cells to Dox-induced apoptosis. Combined Dox + DMPS treatment-induced apoptosis was accompanied by the activation of caspase-8 and caspase-3 as well as PARP cleavage. Cytochrome c and AIF release were also observed in Dox + DMPS-treated HL60/MX2 cells. Pretreatment with z-VAD-fmk markedly prevented caspase-3 activation and moderately suppressed apoptosis, suggesting that Dox + DMPS-induced apoptosis is somewhat (not completely) dependent on caspase. Cytochrome c and AIF release were not affected by pretreatment with z-VAD-fmk. The ROS scavenger NAC efficiently suppressed not only ROS generation, but also caspase-3-mediated PARP cleavage, apoptosis, and release of cytochrome c and AIF, indicating a role of ROS in combined Dox + DMPS treatment-induced apoptotic death signaling. Taken together, these observations suggest that DMPS may be used as a therapeutic agent for overcoming drug-resistance in cancer cells by enhancing drug-induced apoptosis.
Asunto(s)
Factor Inductor de la Apoptosis/metabolismo , Citocromos c/metabolismo , Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Células HL-60/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Esfingosina/análogos & derivados , Acetilcisteína/farmacología , Clorometilcetonas de Aminoácidos/metabolismo , Antibióticos Antineoplásicos/farmacología , Antioxidantes/farmacología , Inhibidores de Caspasas , Caspasas/metabolismo , Supervivencia Celular , Inhibidores de Cisteína Proteinasa/metabolismo , Activación Enzimática , Depuradores de Radicales Libres/farmacología , Humanos , Mitocondrias/metabolismo , Esfingosina/farmacologíaRESUMEN
One main feature of apoptosis is the sequential degradation of the nuclear structure, including the fragmentation of chromatin and caspase-mediated cleavage of various nuclear proteins. Among these proteins is the Nuclear Mitotic Apparatus protein (NuMA) which plays a specific role in the organization of the mitotic spindle. The exact function of NuMA in the interphase nucleus is unknown, but a number of reports have suggested that it may play a role in chromatin organization and/or gene expression. Here we show that upon cleavage in apoptotic cells, the N-terminal cleavage fragment of NuMA is solubilized while the C-terminal fragment remains associated with the condensed chromatin. Using pancaspase inhibitor z-VAD-fmk and caspase-3 deficient MCF-7 cells, we further show that the solubilization is dependent on caspase-mediated cleavage of NuMA. Finally, the silencing of NuMA by RNAi accelerated nuclear breakdown in apoptotic MCF-7 cells. These results suggest that NuMA may provide structural support in the interphase nucleus by contributing to the organization of chromatin.
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Antígenos Nucleares/metabolismo , Apoptosis/fisiología , Núcleo Celular/metabolismo , Silenciador del Gen , Proteínas Asociadas a Matriz Nuclear/metabolismo , Clorometilcetonas de Aminoácidos/metabolismo , Antígenos Nucleares/genética , Caspasa 3/genética , Caspasa 3/metabolismo , Inhibidores de Caspasas , Proteínas de Ciclo Celular , Línea Celular Tumoral , Cromatina/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Humanos , Proteínas Asociadas a Matriz Nuclear/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The alphavirus Semliki Forest virus (SFV) and its derived vectors induce apoptosis in mammalian cells. Here, we show that apoptosis is associated with the loss of mitochondrial membrane potential followed by the activation of caspase-3, caspase-8, and caspase-9. Cell death can be partially suppressed by treatment with the pan-caspase inhibitor zVAD-fmk. To determine the role of SFV structural proteins in cell death, the temporal course of cell death was compared in cells infected with SFV and cells infected with SFV virus replicon particles (VRPs) lacking some or all of the virus structural genes. In the absence of virus structural proteins, cell death was delayed. The endoplasmic reticulum (ER) stress response, as determined by the splicing of X-box binding protein 1 (XBP1) transcripts and the activation of caspase-12, was activated in virus-infected cells but not in VRP (SFV lacking structural genes)-infected cells. The C/EBP-homologous protein (CHOP) was upregulated by both virus and VRP infections. The virus envelope proteins but not the virus capsid protein triggered ER stress. These results demonstrate that in NIH 3T3 cells, SFV envelope glycoproteins trigger the unfolded protein response of the ER and accelerate apoptotic cell death initiated by virus replicase activity.
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Apoptosis/fisiología , Retículo Endoplásmico/metabolismo , Virus de los Bosques Semliki/metabolismo , Estrés Fisiológico , Clorometilcetonas de Aminoácidos/metabolismo , Animales , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Caspasa 9/metabolismo , Inhibidores de Caspasas , Inhibidores de Cisteína Proteinasa/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Ratones , Células 3T3 NIH , Virus de los Bosques Semliki/genética , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Respuesta de Proteína Desplegada/fisiología , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Previous studies reported that high levels of nitric oxide (NO) induce apoptotic cell death in osteoblasts. We examined molecular mechanisms of cytotoxic injury induced by sodium nitroprusside (SNP), a NO donor, in both glutathione (GSH)-depleted and control U2-OS osteoblasts. Cell viability was reduced by much lower effective concentrations of SNP in GSH-depleted cells compared to normal cells. The data suggest that the level of intracellular GSH is critical in SNP-induced cell death processes of osteoblasts. The level of oxidative stress due to SNP treatments doubled in GSH-depleted cells when measured with fluorochrome H2DCFDA. Pretreatment with the NO scavenger PTIO preserved the viability of cells treated with SNP. Viability of cells treated with SNP was recovered by pretreatment with Wortmannin, an autophagy inhibitor, but not by pretreatment with zVAD-fmk, a pan-specific caspase inhibitor. Large increases of LC3-II were shown by immunoblot analysis of the SNP-treated cells, and the increase was blocked by pretreatment with PTIO or Wortmannin; this implies that under GSH-depleted conditions SNP induces different molecular signaling that lead to autophagic cell death. The ultrastructural morphology of SNP-treated cells in transmission electron microscopy showed numerous autophagic vacuoles. These data suggest NO produces oxidative stress and cellular damage that culminate in autophagic cell death of GSH-depleted osteoblasts.
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Glutatión/metabolismo , Nitroprusiato/toxicidad , Osteoblastos/citología , Osteoblastos/fisiología , Clorometilcetonas de Aminoácidos/metabolismo , Apoptosis/fisiología , Autofagia/fisiología , Muerte Celular/fisiología , Línea Celular Tumoral , Supervivencia Celular/fisiología , Células Cultivadas , Humanos , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico , Osteoblastos/ultraestructura , Estrés Oxidativo/fisiología , Sarcoma , Sales de Tetrazolio/metabolismo , Tiazoles/metabolismoRESUMEN
Matriptase is a transmembrane serine protease that is strongly expressed in epithelial cells. The single-chain zymogen of matriptase is considered to have inherent activity, leading to its own activation (i.e. conversion to the disulphide-linked-two-chain form by cleavage after Thr-Lys-Gln-Ala-Arg614). Also, there is growing evidence that the activation of zymogen occurs at the cell surface and in relation to the acidification and lowering of ionic strength within cell-surface microenvironments. The present study aimed to provide evidence for the involvement of zymogen activity in its activation in physiologically relevant cellular contexts. For this purpose, the activity of a pseudozymogen form of recombinant matriptase (HL-matriptase zymogen) was examined using acetyl-l-Lys-l-Thr-l-Lys-l-Gln-l-Leu-l-Arg-4-methyl-coumaryl-7-amide as a substrate. HL-matriptase zymogen exhibited optimal activity toward the substrate pH approximately 6.0. The substrate hydrolysis at the pH value was hardly detected when NaCl was present at a concentration of 145 mM. In a buffer of pH 6.0 containing 5 mM NaCl, the activity of HL-matriptase zymogen was only approximately 30-times lower than that of the respective two-chain form. These findings suggest that the in vivo activation of matriptase zymogen occurs via a mechanism involving the zymogen activity.