RESUMEN
Objective- PAR4 (protease-activated receptor 4), one of the thrombin receptors in human platelets, has emerged as a promising target for the treatment of arterial thrombotic disease. Previous studies implied that thrombin exosite II, known as a binding site for heparin, may be involved in thrombin-induced PAR4 activation. In the present study, a heparin octasaccharide analog containing the thrombin exosite II-binding domain of heparin was chemically synthesized and investigated for anti-PAR4 effect. Approach and Results- PAR4-mediated platelet aggregation was examined using either thrombin in the presence of a PAR1 antagonist or γ-thrombin, which selectively activates PAR4. SCH-28 specifically inhibits PAR4-mediated platelet aggregation, as well as the signaling events downstream of PAR4 in response to thrombin. Moreover, SCH-28 prevents thrombin-induced ß-arrestin recruitment to PAR4 but not PAR1 in Chinese Hamster Ovary-K1 cells using a commercial enzymatic complementation assay. Compared with heparin, SCH-28 is more potent in inhibiting PAR4-mediated platelet aggregation but has no significant anticoagulant activity. In an in vitro thrombosis model, SCH-28 reduces thrombus formation under whole blood arterial flow conditions. Conclusions- SCH-28, a synthetic small-molecular and nonanticoagulant heparin analog, inhibits thrombin-induced PAR4 activation by interfering with thrombin exosite II, a mechanism of action distinct from other PAR4 inhibitors that target the receptor. The characteristics of SCH-28 provide a new strategy for targeting PAR4 with the potential for the treatment of arterial thrombosis.
Asunto(s)
Antitrombinas/farmacología , Heparina/química , Oligosacáridos/farmacología , Agregación Plaquetaria/efectos de los fármacos , Receptores de Trombina/antagonistas & inhibidores , Animales , Antitrombinas/síntesis química , Células CHO , Señalización del Calcio/efectos de los fármacos , Simulación por Computador , Cricetulus , Evaluación Preclínica de Medicamentos , Humanos , Técnicas In Vitro , Modelos Moleculares , Proteínas Recombinantes/efectos de los fármacos , Trombina/farmacología , Trombosis/prevención & controlRESUMEN
Prostratin, a non-tumor promoting 12-deoxyphorbol ester, has been reported as a protein kinase C (PKC) activator and is shown to have anti-proliferative activity in certain cancer cell types. Here we show that GRC-2, a prostratin analogue isolated from Euphorbia grandicornis, is ten-fold more potent than prostratin for inhibiting the growth of human non-small cell lung cancer (NSCLC) A549 cells. Flow cytometry assay revealed that GRC-2 and prostratin inhibited cell cycle progression at the G2/M phase and induced apoptosis. The cytotoxic effect of GRC-2 and prostratin was accompanied by activation and nuclear translocation of PKC-δ and PKD as well as hyperactivation of extracellular signal-related kinase (ERK). Knockdown of either PKC-δ, PKD or ERK significantly protected A549 cancer cells from GRC-2- and prostratin-induced growth arrest as well as apoptosis. Taken together, our results have shown that prostratin and a more potent analogue GRC-2 reduce cell viability in NSCLC A549 cells, at least in part, through activation of the PKC-δ/PKD/ERK pathway, suggesting the potential of prostratin and GRC-2 as anticancer agents.
Asunto(s)
Apoptosis/efectos de los fármacos , Carcinógenos/farmacología , Proliferación Celular/efectos de los fármacos , Ésteres del Forbol/farmacología , Transducción de Señal/efectos de los fármacos , Células A549 , Carcinógenos/química , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Ésteres del Forbol/química , Proteína Quinasa C/metabolismo , Proteína Quinasa C-delta/metabolismoRESUMEN
The capacity of cancer cells to resist detachment-induced apoptosis, i.e. anoikis, as well as anchorage-independent growth are crucial prerequisites for tumor metastasis. Therefore, agents interfering these properties may provide novel anti-metastatic strategies. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, is known as a potent chemopreventive agent, but its effect on anoikis resistance has not been investigated. In this study, two non-small cell lung cancer (NSCLC) cell lines, A549 and CL1-5 cells, were treated with SFN under either suspension or adhesion conditions. SFN exhibited more potent cytotoxicity against suspending rather than adherent cancer cells. The selective cytotoxicity was due to the induction of anoikis, as evident by chromatin condensation, Annexin V binding, and activation of the mitochondrial apoptotic pathway. SFN also inhibited NSCLC cell to form spherical colonies, suggesting that anchorage-independent growth was prevented by SFN. Consistently, SFN treatment led to inactivation of FAK and Akt, down-regulation of ß-catenin, and up-regulation of the cyclin-dependent kinase inhibitor p21. Because A549 cells with wild-type p53 are more sensitive to SFN than p53-mutant CL1-5 cells, p53 dependency of SFN responses were determined in p53-knockdown A549 cells. Knockdown of p53 attenuated the ability of SNF to inhibit anoikis resistance and sphere formation in A549 cancer cells, suggesting that the presence of p53 in NSCLC cancer cells is involved in the sensitivity to SFN. These results provide new insight into mechanisms underlying the chemopreventive ability of SFN and suggest a potential benefit of SFN to interfere with tumor metastasis.
Asunto(s)
Anticarcinógenos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Isotiocianatos/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Anoicis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/genética , Sulfóxidos , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Human platelets contain conventional (α and ß) and novel isoforms of PKC (δ and θ), and PKC activation can result in platelet aggregation and secretion reaction that are important for thrombus formation. Several tumor-promoting Euphorbiaceae diterpenes are known to act as direct activators of PKC, but many types of such diterpenes have not been studied as platelet stimulators. In the present study, two new and five known phorbol esters were isolated from Euphorbia grandicornis. Two of the isolated phorbol esters together with compounds representing ingenane, jatrophane, and myrsinane structural types were studied on PKC activation and platelet stimulation. The investigated phorbol esters and ingenane esters induced blood platelet aggregation and ATP secretion. PKC activation was demonstrated by inducing membrane translocation of PKCs, phosphorylation of PKC substrates, and activation of PKC signaling pathways. The PKC-activating effect of the compounds correlated well with their efficacy to cause platelet stimulation. Moreover, by using an isoform-specific PKC inhibitor, it was found that besides conventional PKCs novel PKCs also play a positive role in platelet activation caused by phorbol/ingenane esters, especially in regulating platelet aggregation. The present results suggest that platelets afford a useful model for studying PKC activators of natural origin or their chemical derivatives.
Asunto(s)
Plaquetas/efectos de los fármacos , Diterpenos/aislamiento & purificación , Diterpenos/farmacología , Euphorbia/química , Proteína Quinasa C/efectos de los fármacos , Diterpenos/química , Humanos , Hungría , Estructura Molecular , Fosforilación , Componentes Aéreos de las Plantas/química , Agregación Plaquetaria/efectos de los fármacos , Isoformas de Proteínas , Proteína Quinasa C/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Abundant clinical evidences indicate that up-regulation of several cathepsins in many human cancers is correlated with malignant progression and poor patient prognosis. In addition, a decrease in catalase activity or accumulation of hydrogen peroxide correlates with cancer metastasis. Recent studies indicate that cathepsin activation and expression can be modulated via H2O2 treatment. However, the actual relationship between catalase and cathepsins is not yet fully understood. In the present study, we found that catalase expression (or activity) was higher, while intracellular and extracellular Cat S, Cat L, and Cat K activities were lower in the non-invasive CL1-0 cells compared to the highly invasive CL1-5 cells. After CL1-0 cells were transfected with catalase-shRNA, the corresponding ROS (H2O2) level and Cat S, Cat L, or Cat K expression (or activity) was up-regulated, accompanied by an increase in cell migration and invasion. On the other hand, ROS (H2O2) level, cathepsin S, L, and K activities, cell migration and invasion were decreased in catalase-overexpressed CL1-5 cells. It is suggested that catalase may regulate cathepsin activity by controlling the production of ROS (H2O2), leading to variation in migration and invasion ability of lung cancer cells.
Asunto(s)
Catalasa/metabolismo , Catepsina K/metabolismo , Catepsina L/metabolismo , Catepsinas/metabolismo , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Catalasa/genética , Catepsina K/genética , Catepsina L/genética , Catepsinas/genética , Línea Celular Tumoral , Movimiento Celular/genética , Humanos , Peróxido de Hidrógeno/metabolismo , Invasividad Neoplásica , Metástasis de la Neoplasia , Interferencia de ARN , ARN Interferente Pequeño , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia ArribaRESUMEN
Elevated cathepsin S (Cat S) level is correlated with higher migration ability in tumor cells. This study investigates the inhibitory effect of novel synthetic α-ketoamide compounds on cathepsin activity and cancer cell migration. The effect of several α-ketoamide compounds on the activity of recombinant cathepsins (Cat S, Cat L and Cat K) was examined. Two highly metastatic cancer cell lines were incubated with three Cat S-specific compounds (6n, 6 w and 6r) to analyze their effect on cellular Cat S activity and cell migration. At a 100 nM concentration, compounds 6n, 6r and 6 w effectively inhibited Cat S activity. Cat S activity and cell migration were significantly reduced in CL1-3 cells after treatment with either 6n or 6 w at 5 µM. Similar results were also obtained when A2058 cells were treated with 6n. These results highlight the therapeutic potential of α-ketoamide compounds, especially 6n and 6 w, to prevent or delay cancer metastasis.
Asunto(s)
Amidas/farmacología , Antineoplásicos/farmacología , Catepsinas/antagonistas & inhibidores , Movimiento Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Amidas/síntesis química , Amidas/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Catepsinas/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Estructura Molecular , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
In this study, a series of 2- and/or 3-substituted juglone derivatives were designed and synthesized. Among them, 9, 18, 22, 30, and 31 showed stronger inhibition activity against cell surface PDI or recombinant PDI and higher inhibitory effects on U46619- and/or collagen-induced platelet aggregation than juglone. The glycosylated derivatives 18 and 22 showed improved selectivity for inhibiting the proliferation of multiple myeloma RPMI 8226 cells, and the IC50 values reached 61 and 48 nM, respectively, in a 72 h cell viability test. In addition, 18 and 22 were able to prevent tumor cell-induced platelet aggregation and platelet-enhanced tumor cell proliferation. The molecular docking showed the amino acid residues Gln243, Phe440, and Leu443 are important for the compound-protein interaction. Our results reveal the potential of juglone derivatives to serve as novel antiplatelet and anticancer dual agents, which are available to interrupt platelet-cancer interplay through covalent binding to PDI catalytic active site.
Asunto(s)
Antineoplásicos , Naftoquinonas , Neoplasias , Humanos , Proteína Disulfuro Isomerasas , Simulación del Acoplamiento Molecular , Plaquetas/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/metabolismo , Neoplasias/metabolismoRESUMEN
There is growing evidence of the importance of protease-activated receptor 4 (PAR4), one of thrombin receptors, as a therapeutic target in thrombotic cardiovascular diseases. In the present study, we utilized ligand-based virtual screening, bioassay, and structure-activity relationship study to discover PAR4 antagonists with new chemical scaffolds from natural origin, and examined their application as antiplatelet agents. By using these approaches, we have identified a flavonoid, 7, 4'-dimethoxy-3-hydroxyflavone, that exhibits anti-PAR4 activity. 7, 4'-Dimethoxy-3-hydroxyflavone inhibited PAR4-mediated human platelet aggregation, GPIIb/IIIa activation, and P-selectin secretion. Also, it inhibited PAR4 downstream signaling pathways, including Ca2+/protein kinase C, Akt, and MAP kinases ERK and p38, in human platelets, and suppressed PAR4-mediated ß-arrestin recruitment in CHO-K1 cells exogenously expressed human PAR4. In a microfluidic system, 7, 4'-dimethoxy-3-hydroxyflavone reduced thrombus formation on collagen-coated chambers at an arterial shear rate in recalcified whole blood. Furthermore, mice treated with 7, 4'-dimethoxy-3-hydroxyflavone were significantly protected from FeCl3-induced carotid arterial occlusions, without significantly affecting tail bleeding time. In conclusion, 7, 4'-dimethoxy-3-hydroxyflavone represents a new class of nature-based PAR4 antagonist, it shows effective in vivo antithrombotic properties with less bleeding tendency, and could be a potential candidate for developing new antiplatelet agents.
Asunto(s)
Inhibidores de Agregación Plaquetaria , Trombosis , Animales , Humanos , Ratones , Plaquetas , Fibrinolíticos/metabolismo , Flavonoides/metabolismo , Flavonoides/farmacología , Flavonoides/uso terapéutico , Agregación Plaquetaria , Inhibidores de Agregación Plaquetaria/metabolismo , Receptores de Trombina/metabolismo , Trombina/metabolismo , Trombosis/tratamiento farmacológico , Trombosis/metabolismoRESUMEN
Inflammation in the tumor microenvironment is positively correlated with cancer progression and metastasis as well as the risk of thromboembolism in lung cancer patients. Here we show, in human non-small cell lung cancer (NSCLC) cell lines, the master inflammatory cytokine tumor necrosis factor (TNF-α) induced tissue factor expression and procoagulant activity, and these effects were potently inhibited by 4ß-hydroxywithanolide E (4HW), a natural compound isolated from Physalis peruviana. Furthermore, combination of 4HW and TNF-α caused synergistic cytotoxicity against NSCLC cells by inducing caspase-dependent apoptosis. The underlying mechanism by which 4HW reverses the procoagulant effect of TNF-α but enhances its cytotoxic effect appears to be due to inhibition of NF-κB, which is a key switch for both inflammation-induced coagulation and cell survival. Our results suggest that 4HW may have a potential application for treating inflammation-derived cancer progression and cancer-associated hypercoagulable state.
Asunto(s)
Antineoplásicos/farmacología , Coagulación Sanguínea/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Physalis/química , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Witanólidos/farmacología , Células A549/efectos de los fármacos , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Línea Celular Tumoral , Humanos , Microscopía Fluorescente , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Uridine monophosphate (UMP) kinase converts UMP to the corresponding UDP in the presence of metal ions and ATP and is allosterically regulated by nucleotides such as UTP and GTP. Although the UMP kinase reported to date is Mg(2+)-dependent, we found in this study that the UMP kinase of Helicobacter pylori had a preference for Mn(2+) over Mg(2+), which may be related to a conformational difference between the Mn(2+)-bound and Mg(2+)-bound UMP kinase. Similar to previous findings, the UMP kinase activity of H. pylori UMP kinase was inhibited by UTP and activated by GTP. However, a relatively low GTP concentration (0.125 mM) was required to activate H. pylori UMP kinase to a level similar to other bacterial UMP kinases using a higher GTP concentration (0.5 mM). In addition, depending on the presence of either Mg(2+) or Mn(2+), a significant difference in the level of GTP activation was observed. It is therefore hypothesized that the Mg(2+)-bound and Mn(2+)-bound H. pylori UMP kinase may be activated by GTP through different mechanisms.
Asunto(s)
Proteínas Bacterianas/metabolismo , Helicobacter pylori/enzimología , Manganeso/metabolismo , Nucleósido-Fosfato Quinasa/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Guanosina Trifosfato/metabolismo , Helicobacter pylori/genética , Magnesio/metabolismo , Datos de Secuencia Molecular , Nucleósido-Fosfato Quinasa/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Triple-negative breast cancer (TNBC) represents a clinical challenge because it lacks sensitivity to hormone therapy or other available molecule-targeted agents. In addition, TNBC frequently exhibits over-activation of the PI3K/Akt survival pathway that can contribute to chemotherapy resistance. 4ß-Hydroxywithanolide E (4-HW) and withaferin A (WA) are two withanolides from Solanaceae plants that exhibit promising anticancer activity in vitro and in vivo. PURPOSE: The aim of this study is to investigate and compare the effects of 4-HW and WA on TNBC cells and underling mechanisms. STUDY DESIGN/METHODS: The anticancer effects of 4-HW and WA were evaluated by cell viability, cell cycle arrest, and apoptosis assays. PI3K/Akt signaling and the expression of survivin, Bcl-2 family proteins and cyclin-dependent kinase inhibitors were evaluated by Western blot. The role of PI3K/Akt signaling in the withanolides-induced anticancer effects was examined by using a PI3K inhibitor and overexpression of a constitutively active form of Akt. RESULTS: In TNBC MDA-MB-231 cells, 4-HW and WA displayed different kinetic effect on cell availability. Cell cycle analysis revealed that 4-HW induced the G1-phase arrest while WA caused the G2/M-phase block. Both withanolides induced apoptosis, but WA also caused necrosis. 4-HW inhibited the PI3K/Akt pathway and survivin expression as well as up-regulated the cyclin-dependent kinase inhibitors p21 and p27. In contrast, WA is a more potent inhibitor of Hsp90 and elicited Akt activation at low doses but inhibited Akt signaling at higher doses by depleting the Akt protein. The PI3K inhibitor LY294002 mimicked the effects of 4-HW and potentiated the cytotoxic activity of WA. In contrast, overexpressing a constitutively active form of myristoylated Akt rescue cancer cells from 4-HW-induced cell death. CONCLUSION: The withanolides 4-HW and WA potently inhibit the viability of TNBC cells through induction of cell cycle arrest and apoptosis/necrosis. The PI3K/Akt pathway plays distinct roles in cancer cells respond to 4-HW and WA. These results suggest the potential applications of the withanolides for the treatment of TNBC.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Witanólidos/farmacología , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Solanaceae/química , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Clustered DNA damage other than double-strand breaks (DSBs) can be detrimental to cells and can lead to mutagenesis or cell death. In addition to DSBs induced by ionizing radiation, misrepair of non-DSB clustered damage contributes extra DSBs converted from DNA misrepair via pathways for base excision repair and nucleotide excision repair. This study aimed to quantify the relative biological effectiveness (RBE) when DSB induction and conversion from non-DSB clustered damage misrepair were used as biological endpoints. The results showed that both linear energy transfer (LET) and indirect action had a strong impact on the yields for DSB induction and conversion. RBE values for DSB induction and maximum DSB conversion of helium ions (LET = 120 keV/µm) to (60)Co gamma rays were 3.0 and 3.2, respectively. These RBE values increased to 5.8 and 5.6 in the absence of interference of indirect action initiated by addition of 2-M dimethylsulfoxide. DSB conversion was â¼1-4% of the total non-DSB damage due to gamma rays, which was lower than the 10% estimate by experimental measurement. Five to twenty percent of total non-DSB damage due to helium ions was converted into DSBs. Hence, it may be possible to increase the yields of DSBs in cancerous cells through DNA repair pathways, ultimately enhancing cell killing.