RESUMEN
Phosphatidylinositol 3-kinases (PI3Ks) mediate intracellular signal transduction. Aberrant PI3K signaling is associated with oncogenesis and disease progression in solid tumors and hematologic malignancies. Idelalisib (1), a first-in-class PI3Kδ inhibitor for the treatment of hematologic malignancies, was developed, but its sales were limited by black box warnings due to unexpected adverse effects. Therefore, to overcome these adverse events, various quinazolinone derivatives were synthesized and evaluated in vitro based on their inhibitory activity against the PI3K enzyme and the viability of cell lines such as MOLT and SUDHL. Among them, 6f (IC50 = 0.39 nM) and 6m (IC50 = 0.09 nM) showed excellent enzyme activity, and 6m displayed an approximately four-fold higher selectivity for PI3Kγ/δ compared with Idelalisib (1). Furthermore, in vivo PK experiments with 6f and 6m revealed that 6f (AUClast = 81.04 h*ng/mL, Cmax = 18.34 ng/mL, Tmax = 0.5 h, t1/2 = 10.2 h in 1 mpk dose) had improved PK compared with 1. Finally, further experiments will be conducted with 6f selected as a candidate, and the potential for it to be developed as a treatment with good efficacy for hematologic malignancies will be determined.
Asunto(s)
Antineoplásicos/farmacología , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Neoplasias Hematológicas/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Purinas/farmacología , Quinazolinonas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Inhibidores de las Quinasa Fosfoinosítidos-3/síntesis química , Inhibidores de las Quinasa Fosfoinosítidos-3/química , Purinas/química , Quinazolinonas/síntesis química , Quinazolinonas/química , Relación Estructura-ActividadRESUMEN
Here, we explored the anti-tumor efficacy of a cyclic pentadepsipeptide, N-methylsansalvamide (MSSV), in bladder cancer. MSSV inhibited the proliferation of both bladder cancer 5637 and T24 cells, which was attributed to the G1-phase cell cycle arrest, apoptosis induction, and alteration of mitogen-activated protein kinases (MAPKs) and protein kinase b (AKT) signaling pathways. Additionally, the treatment of bladder cancer cells with MSSV suppressed migratory and invasive potential via the transcription factor-mediated expression of matrix metalloproteinase 9 (MMP-9). MSSV abrogated vascular endothelial growth factor (VEGF)-induced angiogenic responses in vitro and in vivo. Furthermore, our result showed the potent anti-tumor efficacy of MSSV in a xenograft mouse model implanted with bladder cancer 5637 cells. Finally, acute toxicity test data obtained from blood biochemical test and liver staining indicated that the oral administration of MSSV at 2000 mg/kg caused no adverse cytotoxic effects. Our preclinical data described the potent anti-angiogenic and anti-tumor efficacy of MSSV and showed no signs of acute toxicity, thereby suggesting the putative potential of oral MSSV as a novel anti-tumor agent in bladder cancer treatment.
RESUMEN
Carnosine, a naturally producing dipeptide, exhibits various beneficial effects. However, the possible role of carnosine in vascular disorders associated with pathological conditions, including proliferation and migration of vascular smooth muscle cells (VSMCs), largely remains unrevealed. Here, we investigated the regulatory role and mechanism of carnosine in platelet-derived growth factor (PDGF)-induced VSMCs. Carnosine inhibited the proliferation of PDGF-induced VSMCs without any cytotoxic effects. Carnosine treatment also induced G1-phase cell cycle arrest by causing a p21WAF1-mediated reduction in the expression of both cyclin-dependent kinases (CDKs) and cyclins in PDGF-treated VSMCs. Carnosine treatment suppressed c-Jun N-terminal kinase (JNK) phosphorylation in PDGF-stimulated signaling. Additionally, carnosine significantly prevented the migration of VSMCs exposed to PDGF. Carnosine abolished matrix metalloproteinase (MMP)-9 activity via reduced transcriptional binding activity of NF-κB, Sp-1, and AP-1 motifs in PDGF-treated VSMCs. Moreover, using aortic assay ex vivo, it was observed that carnosine addition attenuated PDGF-stimulated sprout outgrowth of VSMCs. Taken together, these results demonstrated that carnosine impeded the proliferation and migration of PDGF-stimulated VSMCs by regulating cell cycle machinery, JNK signaling, and transcription factor-mediated MMP-9 activity as well as prevented ex vivo sprout outgrowth of blood vessels. Thus, carnosine may be a potential candidate for preventing vascular proliferative disease.
Asunto(s)
Carnosina/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/efectos adversos , Animales , Supervivencia Celular/efectos de los fármacos , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/metabolismo , FN-kappa B/metabolismo , Fosforilación , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Cicatrización de HeridasRESUMEN
BACKGROUND: Numerous studies have focused on solvent extracts from locust trees (Gleditsia spp.), which contain diverse bioactive components including saponins, flavonoids, and alkaloids. However, because of the undefined nature of such phytochemicals, their clinical application as chemotherapeutic agents has often been limited. PURPOSE: This study aimed to evaluate the anti-oncogenic activity of triacanthine, an alkaloid obtained from Gleditsia triacanthos L. STUDY DESIGN: The anti-oncogenicity of triacanthine in vitro was evaluated via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell-counting kit-8 assay (CCK-8 assay), flow cytometry, imunoblot, migration and invasion assays, zymography, and electrophoretic mobility shift assay in the human bladder carcinoma cell line EJ. The in vivo efficacy of triacanthine was evaluated via oral administration to EJ-xenografted BALB/c nude mice. To identify the side effects of triacanthine, cisplatin was also administered and an acute toxicity test was performed. RESULTS: Triacanthine significantly inhibited EJ cell proliferation (IC50 600⯵M). Flow cytometry analysis revealed that cells were arrested in the G1 phase, and apoptotic cells accumulated in sub-G1 phase in a dose-dependent manner. Triacanthine inhibited the G1-S transition by deterring complex formation between cyclin-dependent kinases and cyclins, thereby up-regulating cell cycle inhibitors p21WAF1 and p27KIP1. In addition, triacanthine induced a caspase-dependent extrinsic pathway of apoptosis and autophagy. Early responsive kinases, extracellular signal-regulated kinase (ERK) and Janus kinase (JNK) were up-regulated by triacanthine. Triacanthine-mediated inhibition of the migratory and invasive potential of EJ cells was attributed to reduction of matrix metalloproteinase (MMP)-9 due to suppression of binding activities of the transcription factors activator protein (AP)-1, specificity protein (Sp)-1, and nuclear factor (NF)-κB. In an in vivo study, triacanthine significantly limited growth of xenografted tumors. Interestingly, while cisplatin resulted in significant weight loss after a 5-mg/kg dose, triacanthine did not cause weight loss, behavioral abnormalities, altered biochemical parameters, or tissue staining. A single oral dose acute-toxicity test (triacanthine 2,000â¯mg/kg) produced no adverse cytotoxic effects via blood biochemical tests and tissue-organ staining. CONCLUSION: To our knowledge, this is the first systematic evaluation of the anti-oncogenic activity of triacanthine. Therefore, we believe that our findings may guide the development of novel chemotherapeutic agents for bladder cancers.
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Alcaloides/farmacología , Antineoplásicos/farmacología , Gleditsia/química , Fitoquímicos/farmacología , Purinas/farmacología , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Quinasas Janus/efectos de los fármacos , Quinasas Janus/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones DesnudosRESUMEN
Hyper-proliferation and migration of vascular smooth muscle cells (VSMCs) are closely associated with atherosclerosis. Recently, the flavonol morin has been reported to exhibit potent anti-oxidant and anti-inflammatory activities. Therefore, we investigated molecular mechanisms of morin in VSMCs stimulated by PDGF. Morin effectively inhibited PDGF-stimulated proliferation of VSMCs through a G1 cell-cycle arrest, leading to down-regulation of CDK2, CDK4, cyclin D1, and cyclin E proteins. Interestingly, PDGF markedly down-regulated p27KIP1 protein expression; however, morin treatment restored the p27KIP1expression to the basal level. Morin did not affect phosphorylation of MAPKs (ERK, p38, and JNK); however, phosphorylation of AKT was dramatically suppressed by morin in PDGF-stimulated VSMCs. Using the PI3K inhibitor, LY294002, we revealed that AKT is a key regulator in the inhibitory mechanism of morin against PDGF-induced proliferation of VSMCs. Morin disturbed migratory and invasive potential of VSMCs via suppression of matrix metalloproteinase-9 (MMP-9) activity. Using electrophoretic mobility shift assays, we verified that NF-κB, AP-1, and Sp-1 transcription factors are implicated in the mode of action of morin, which suppresses the MMP-9 activity in PDGF-induced VSMCs. Based on the results, we believe that morin may be a potential therapeutic agent for atherosclerosis without negative side effect.
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Proliferación Celular , Movimiento Celular , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Flavonoides , Metaloproteinasa 9 de la Matriz , Músculo Liso Vascular , Miocitos del Músculo Liso , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-aktRESUMEN
Hydrangenol is a dihydroisocoumarin that is mainly obtained from Hydrangea macrophylla. Recently, hydrangenol has garnered attention since several studies have reported that it has anti-inflammatory, anti-allergic, anti-diabetic, and anti-malarial activities. However, there have been few studies on the effect of hydrangenol on oncogenesis. In this study, we evaluated the anti-cancer activity of hydrangenol against the EJ bladder cancer cell line. Hydrangenol significantly inhibited the proliferation of EJ cells in a dose-dependent manner with an IC50 of 100 µM. Flow cytometry and immunoblotting experiments indicated that EJ cells were arrested in the G1-phase of the cell cycle and showed reduced expression of CDK2, CDK4, cyclin D1, and cyclin E mediated via the upregulation of p21WAF1. Hydrangenol increased the phosphorylation of p38 MAPK without affecting the phosphorylation of ERK and JNK. In addition, hydrangenol significantly inhibited the migratory and invasive activities of EJ cells by suppressing the enzymatic activity of MMP-9. Electrophoretic mobility shift assays suggested that the inhibition of MMP-9 activity by hydrangenol was attributable to its suppression of the Sp-1 transcription factor binding activity. This study is the first report on the mode of action of hydrangenol as an inhibitor of bladder cancer. We believe that these results provide novel insights that could aid the development of hydrangenol-based chemotherapeutic agents.
RESUMEN
BACKGROUND: Although the diverse biological properties of nanoparticles have been studied intensively, research into their mechanism of action is relatively rare. In this study, we investigated the molecular mechanisms of the anticancer activity of heterometallic Au@Pt-nanoseeds (NSs) against bladder cancers. MATERIALS AND METHODS: Mode of action of Au@Pt-NSs was investigated through MTT assay, flow cytometry analysis, Western immunoblots, real-time qPCR, wound-healing migration and invasion assays, zymography, and electrophoretic mobility shift assay (EMSA). RESULTS: Treatment with Au@Pt-NSs significantly inhibited the proliferation of EJ cells in a dose-dependent manner by inducing G1 phase cell cycle arrest. Among the regulators associated with the G1 cell cycle phase, CDK2, CDK4, cyclin D1, cyclin E, and p21WAF1 were shown to participate in the inhibitory pathways of Au@Pt-NSs. In addition, treatment with Au@Pt-NSs led to upregulation of phospho-p38 MAPK and downregulation of phospho-AKT in EJ cells. Interestingly, Au@Pt-NSs inhibited the migratory and invasive potential of the cells, which was attributed to the suppression of the enzymatic activity of matrix metalloproteinase-9 (MMP-9). Using MMP-9-specific oligonucleotides, we showed that transcription factors such as NF-κB and Sp-1 were responsible for the MMP-9-mediated metastatic potential of EJ cells. CONCLUSION: Au@Pt-NSs significantly limited the progression, migration, and invasion of bladder cancer EJ cells. Our data represent a novel insight into developing cisplatin-like chemotherapeutic reagents with fewer side effects and provide useful information on molecular markers to monitor patients under Au@Pt-NSs-based chemotherapy.
Asunto(s)
Antineoplásicos/farmacología , Oro/farmacología , Platino (Metal)/farmacología , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Antineoplásicos/química , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ciclina D1/metabolismo , Oro/química , Humanos , Metaloproteinasa 9 de la Matriz/metabolismo , FN-kappa B/metabolismo , Nanoestructuras/química , Platino (Metal)/química , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Background: Diallyl trisulfide (DATS), a bioactive sulfur compound in garlic, has been highlighted due to its strong anti-carcinogenic activity. Objective: The current study investigated the molecular mechanism of garlic-derived DATS in cancer cells. Additionally, we explored possible molecular markers to monitoring clinical responses to DATS-based chemotherapy. Design: EJ bladder carcinoma cells were treated with different concentration of DATS. Molecular changes including differentially expressed genes in EJ cells were examined using immunoblot, FACS cell cycle analysis, migration and invasion assays, electrophoresis mobility shift assay (EMSA), microarray, and bioinformatics analysis. Results: DATS inhibited EJ cell growth via G2/M-phase cell cycle arrest. ATM-CHK2-Cdc25c-p21WAF1-Cdc2 signaling cascade, MAPKs, and AKT were associated with the DATS-mediated growth inhibition of EJ cells. DATS-induced inhibition of migration and invasion was correlated with down-regulated MMP-9 via reduced activation of AP-1, Sp-1, and NF-κB. Through microarray gene expression analysis, ANGPTL4, PLCXD1, and MMP3 were identified as candidates of molecular targets of DATS. Introduction of each gene to EJ cells revealed that ANGPTL4 was associated with the DATS-induced inhibition of cell growth, migration, and invasion. Conclusions: ANGPTL4 regulates DATS-mediated inhibition of proliferation, migration, and invasion of EJ cells, and thus, has potential as a prognostic marker for bladder cancer patients.
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We report a pivotal role for IL-5 as an angiogenic activator. IL-5 increased proliferation, migration and colony tube formation in HUVECs associated with the phosphorylation of ERK and AKT/eNOS, and promoted microvessel sprouting from an angiogenesis animal model. The angiogenic effects were confirmed in IL-5-deficient mice and addition of IL-5 antibody. HSP70-1 was identified via expression profiling following IL-5 stimulation. A siRNA knockdown of HSP70-1 suppressed angiogenic responses and eNOS phosphorylation induced by IL-5. HSP70-1 overexpression enhanced IL-5-induced angiogenic responses. In addition, IL-5-induced neo-vascular formation was verified in both HSP70-1 knockout and HSP70-1 transgenic mice. Furthermore, transcription factor AP-1 was a main factor in IL-5-induced HSP70-1 in response to ERK and AKT signaling pathway. Angiogenic responses induced by VEGF had no effect in either HSP70-1 siRNA in vitro or HSP70-1 knockout mice. IL-5-induced angiogenic responses depended on the binding of IL-5Rα. Our data demonstrate that binding of IL-5 to IL-5Rα receptors enhances angiogenic responses by stimulating the expression of HSP70-1 via the eNOS signaling pathway.
Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Interleucina-5/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Transducción de Señal , Animales , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Perfilación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Interleucina-5/deficiencia , Interleucina-5/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Microvasos/efectos de los fármacos , Microvasos/crecimiento & desarrollo , Neovascularización Fisiológica/genética , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacología , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Although recent studies have demonstrated the anti-tumor effects of garlic extract (GE), the exact molecular mechanism is still unclear. In this study, we investigated the molecular mechanism associated with the inhibitory action of GE against bladder cancer EJ cell responses. Treatment with GE significantly inhibited proliferation of EJ cells dose-dependently through G2/M-phase cell cycle arrest. This G2/M-phase cell cycle arrest by GE was due to the activation of ATM and CHK2, which appears to inhibit phosphorylation of Cdc25C (Ser216) and Cdc2 (Thr14/Tyr15), this in turn was accompanied by down-regulation of cyclin B1 and up-regulation of p21WAF1. Furthermore, GE treatment was also found to induce phosphorylation of MAPK (ERK1/2, p38MAPK, and JNK) and AKT. In addition, GE impeded the migration and invasion of EJ cells via inhibition of MMP-9 expression followed by decreased binding activities of AP-1, Sp-1, and NF-κB motifs. Based on microarray datasets, we selected Heat shock protein A6 (HSPA6) as the most up-regulated gene responsible for the inhibitory effects of GE. Interestingly, overexpression of HSPA6 gene resulted in an augmentation effect with GE inhibiting proliferation, migration, and invasion of EJ cells. The augmentation effect of HSPA6 was verified by enhancing the induction of G2/M-phase-mediated ATM-CHK2-Cdc25C-p21WAF1-Cdc2 cascade, phosphorylation of MAPK and AKT signaling, and suppression of transcription factor-associated MMP-9 regulation in response to GE in EJ cells. Overall, our novel results indicate that HSPA6 reinforces the GE-mediated inhibitory effects of proliferation, migration, and invasion of EJ cells and may provide a new approach for therapeutic treatment of malignancies.
Asunto(s)
Antineoplásicos/farmacología , Ajo/química , Proteínas HSP70 de Choque Térmico/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Extractos Vegetales/farmacología , Neoplasias de la Vejiga Urinaria/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quinasa de Punto de Control 2/metabolismo , Humanos , Sistema de Señalización de MAP QuinasasRESUMEN
The pharmacological effects of Rosa hybrida are well known in the cosmetics industry. However, the role of Rosa hybrida in cardiovascular biology had not previously been investigated, to the best of our knowledge. The aim of the present study was to elucidate the effect of water extract of Rosa hybrida (WERH) on plateletderived growth factor (PDGF)-stimulated vascular smooth muscle cells (VSMCs). VSMC proliferation, which was stimulated by PDGF, was inhibited in a non-toxic manner by WERH treatment, which also diminished the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT. Treatment with WERH also induced G1-phase cell cycle arrest, which was due to the decreased expression of cyclins and cyclin-dependent kinases (CDKs), and induced p21WAF1 expression in PDGF-stimulated VSMCs. Moreover, WERH treatment suppressed the migration and invasion of VSMCs stimulated with PDGF. Treatment with WERH abolished the expression of matrix metalloproteinase-9 (MMP-9) and decreased the binding activity of nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and specificity protein 1 (Sp1) motifs in PDGF-stimulated VSMCs. WERH treatment inhibited the proliferation of PDGFstimulated VSMCs through p21WAF1mediated G1-phase cell cycle arrest, by decreasing the kinase activity of cyclin/CDK complexes. Furthermore, WERH suppressed the PDGF-induced phosphorylation of ERK1/2 and AKT in VSMCs. Finally, treatment with WERH impeded the migration and invasion of VSMCs stimulated by PDGF by downregulating MMP-9 expression and a reduction in NF-κB, AP-1 and Sp1 activity. These results provide new insights into the effects of WERH on PDGF-stimulated VSMCs, and we suggest that WERH has the potential to act as a novel agent for the prevention and/or treatment of vascular diseases.
Asunto(s)
Metaloproteinasa 9 de la Matriz/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Extractos Vegetales/farmacología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Rosa/química , Transducción de Señal/efectos de los fármacos , Animales , Puntos de Control del Ciclo Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Masculino , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Extractos Vegetales/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Sprague-DawleyRESUMEN
Esculetin is known to inhibit tumor growth, but its effect in angiogenesis has not been studied. Here, we report the efficacy of esculetin on VEGF-induced angiogenesis. Esculetin treatment inhibited VEGF-induced proliferation and DNA synthesis of HUVECs with no cell toxicity. G1-phase cell-cycle arrest was associated with a decreased expression of cyclins and CDKs via the binding of p27KIP1. Esculetin down-regulated the MMP-2 expression in VEGF-stimulated HUVECs, which suppressed colony tube formation and migration. Esculetin reduced the phosphorylation of VEGFR-2 and the downstream signaling of VEGFR-2, including ERK1/2 and eNOS/Akt pathways. Esculetin suppressed microvessel outgrowth from an aortic ring ex vivo model treated with VEGF, and blocked the VEGF-induced formation of new blood vessels and hemoglobin content in an in vivo Matrigel plug model. Collectively, VEGF-stimulated responses in angiogenesis were inhibited in vitro and in vivo, providing a theoretical basis for effective use against anti-angiogenic therapies.
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Antineoplásicos Fitogénicos , Antioxidantes , Neovascularización Patológica/inducido químicamente , Neovascularización Patológica/tratamiento farmacológico , Fitoterapia , Umbeliferonas/farmacología , Umbeliferonas/uso terapéutico , Factor A de Crecimiento Endotelial Vascular/efectos adversos , Artemisia , Hemoglobinas/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Metaloproteinasa 2 de la Matriz/metabolismo , Fosforilación/efectos de los fármacos , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Ophiopogon japonicus is known to have various pharmacological effects. The present study investigated the effects of an extract of fermented Ophiopogon japonicas (FEOJ) on thrombintreated vascular smooth muscle cells (VSMCs). FEOJ treatment inhibited the proliferation of VSMCs treated with thrombin as indicated by an MTT assay. These inhibitory effects were associated with decreased phosphorylation of AKT, reduced expression of cyclin D1 and increased expression of p27KIP1 in thrombininduced VSMCs. In addition, FEOJ treatment suppressed the thrombinstimulated migration of VSMCs as demonstrated by a woundhealing migration assay. Furthermore, zymographic analyses demonstrated that treatment of FEOJ with VSMCs suppressed the thrombininduced expression of matrix metalloproteinase (MMP)2, which was attributed to the reduction of nuclear factor (NF)κB binding activity. Collectively, these results demonstrated that FEOJ induced p27KIP1 expression, reduced cyclin D1 expression and AKT phosphorylation, and inhibited MMP2 expression mediated by downregulation of NFκB binding activity in thrombintreated VSMCs, which led to growth inhibition and repression of migration. These results supported the use of FEOJ for the prevention of vascular diseases and provided novel insight into the underlying mechanism of action.
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Fermentación , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Ophiopogon/química , Extractos Vegetales/farmacología , Animales , Ciclo Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ciclina D1/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/enzimología , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Sprague-Dawley , TrombinaRESUMEN
Interleukin-16 (IL-16) is a lymphocyte chemoattractant factor well known for its role in immune responses, but its role in vascular disease is unknown. Here, we explored the novel physiological function of IL-16 in vascular smooth muscle cells (VSMCs). The expression of IL-16 and its receptor CD4 was observed in VSMCs. Treatment with IL-16 enhanced the migration and invasion by VSMCs without altering the proliferative potential. IL-16 induced MMP-9 expression via the binding activity of transcription factors NF-κB, AP-1, and Sp-1 motifs in VSMCs. Among the relevant signaling pathways examined, only p38MAPK phosphorylation was significantly stimulated in IL-16-treated VSMCs. Treatment with p38MAPK inhibitor SB203580 prevented the IL-16-induced migration and invasion of VSMCs. SB203580 treatment inhibited the MMP-9 expression and activation of Sp-1 binding in IL-16-treated VSMCs, and siRNA knockdown of CD4 expression blocked the induction of migration, invasion, p38MAPK phosphorylation, MMP-9 expression, and Sp-1 binding activation stimulated by IL-16. The IL-16 induced cell-cycle-inhibitor p21WAF1 expression in VSMCs, but had no effect on the expression levels of other cell-cycle negative regulators. Finally, blockage of p21WAF1 function with specific siRNA abolished the IL-16-induced elevation of migration, invasion, p38MAPK phosphorylation, MMP-9 expression, and Sp-1 binding activation in VSMCs. Taken together, p21WAF1 was required for the induction of p38MAPK-mediated MMP-9 expression via activation of the Sp-1 binding motif, which led to migration and invasion of VSMCs interacting with IL-16/CD4. These results could provide that IL-16 is a new target in the treatment of vascular diseases such as atherosclerosis and re-stenosis.
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Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Interleucina-16/metabolismo , Miocitos del Músculo Liso/fisiología , Animales , Antígenos CD4/metabolismo , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Proliferación Celular , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Imidazoles/farmacología , Interleucina-16/farmacología , Sistema de Señalización de MAP Quinasas , Metaloproteinasa 9 de la Matriz/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/inmunología , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , ARN Interferente Pequeño/genética , Ratas , Factor de Transcripción Sp1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
MicroRNAs (miRs) serve either as oncogenes or tumor-suppressor genes in tumor progression. MicroRNA-20b (miR20b) is known to be involved with the oncomirs of several types of cancers. However, in the present study we describe how miR-20b inhibits the proliferation, migration and invasion of bladder cancer EJ cells. In the present study, miR-20b was downregulated in bladder cancer cell lines, and its overexpression resulted in a significant reduction in the proliferation of EJ cells. In addition, via a bioinformatics approach, we identified cell cycle-regulated genes that are the putative targets of miR-20b. The transfection of miR-20b into EJ cells induced G1 phase cell cycle arrest via the decreased expression of cyclin D1, CDK2 and CDK6 without affecting another G1 phase cell cycle regulator, cyclin E. The cell cycle inhibitor p21WAF1 was upregulated in the miR-20b transfected cells. Moreover, the enforced expression of miR-20b resulted in impaired wound-healing migration and invasion in the EJ cells. Based on our target prediction analysis of miRs, we confirmed that miR-20b overexpression strongly impedes MMP-2 expression via suppressive activation of the Sp-1 binding motif, an important transcription factor present in the MMP-2 promoter. Herein, we report the novel concept that miR-20b exerts a suppressive effect on both cell cycle-modulated proliferation and MMP-2-mediated migration and invasion in bladder cancer EJ cells.
Asunto(s)
Puntos de Control del Ciclo Celular/genética , Metaloproteinasa 2 de la Matriz/genética , MicroARNs/genética , Neoplasias de la Vejiga Urinaria/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Ciclina D1/biosíntesis , Quinasa 6 Dependiente de la Ciclina/biosíntesis , Regulación Neoplásica de la Expresión Génica , Humanos , Invasividad Neoplásica/genética , Transfección , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
The use of recombinant human erythropoietin (rHuEpo) can lead to hypertrophy and hyperplasia, and has induced the proliferation of vascular smooth muscle cells (VSMCs). The effect of the EPO gene in the migration and invasion of VSMCs remains unclear. In this study, overexpression of the EPO gene increased the DNA synthesis and phosphorylation of ERK1/2 and p38MAPK in VSMCs. In addition, EPO gene expression induced the migration and invasion of VSMCs via the expression of MMP-9 by the activation of NF-κB and AP-1 binding. A blockade of p38MAPK by specific p38MAPK inhibitor SB203580 led to a suppression of the increased DNA synthesis, migration, and invasion of VSMCs that was induced by the EPO gene. SB203580 treatment blocked the increased expression of MMP-9 through the binding activity of AP-1. Transfection of the EPO gene with VSMCs was associated with the up-regulation of cyclin D1/CDK4, cyclin E/CDK2, and p21WAF1, and with the down-regulation of p27KIP1. The specific suppression of p21WAF1 expression by siRNA rescued the enhancement of DNA synthesis via the phosphorylation of p38MAPK and the increase in migration and invasion through AP-1-mediated MMP-9 expression in EPO gene transfectants. These novel findings demonstrate that p21WAF1 regulates the proliferation, migration and invasion of VSMC induced by EPO gene.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Eritropoyetina/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Factor de Transcripción AP-1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ciclina E/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Eritropoyetina/genética , Humanos , Imidazoles/farmacología , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , FN-kappa B/metabolismo , Proteínas Oncogénicas/metabolismo , Fosforilación/efectos de los fármacos , Piridinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Erythropoietin (EPO) is a cytokine that modulates the production of red blood cells. Previous studies have contradicted the assumed role of EPO in tumor cell proliferation. In the present study, we investigated the effect of EPO in the proliferation, migration and invasion that is involved in the signaling pathways and cell-cycle regulation of bladder cancer 5637 cells. The results showed that an overexpression of the EPO gene has a potent stimulatory effect on DNA synthesis, migration and invasion. EPO gene expression increased the expression of matrix metalloproteinase (MMP)-9 via the binding activity of NF-κB, AP-1 and Sp-1 in 5637 cells. The transfection of 5637 cells with the EPO gene induced the phosphorylation of ERK1/2. Treatment with ERK1/2 inhibitor U0126 significantly inhibited the increased proliferation, migration and invasion of EPO gene-transfected cells. U0126 treatment suppressed the induction of MMP-9 expression through NF-κB binding activity in EPO gene transfectants. In addition, EPO gene expression was correlated with the upregulation of cyclins/CDKs and the upregulation of the CDK inhibitor p21WAF1 expression. Finally, the inhibition of p21WAF1 function by siRNA blocked the proliferation, migration, invasion and phosphorylation of ERK1/2 signaling, as well as MMP-9 expression and activation of NF-κB in EPO gene-transfected cells. These novel findings suggest that the molecular mechanisms of EPO contribute to the progression and development of bladder tumors.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Eritropoyetina/metabolismo , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Butadienos/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Eritropoyetina/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Invasividad Neoplásica , Nitrilos/farmacología , Fosforilación , Neoplasias de la Vejiga Urinaria/genéticaRESUMEN
Interleukin-15 (IL-15) functions as a key regulator for the proliferation, differentiation, and activation of lymphocytes. However, the role of IL-15 in cancer biology is not yet understood. In the present study, IL-15 treatment stimulated the wound-healing migration and invasion of bladder cancer 5637 cells, without altering the proliferation of the cells. Treatment of 5637 cells with IL-15 resulted in the promotion of the MMP-9 expression and the activation of NF-κB binding, which is a functional transcription factor that activates MMP-9 expression. In addition, IL-15 induced the activation of ERK1/2. ERK inhibitor U0126 suppressed the migration, invasion, MMP-9 expression, and NF-κB binding activity in IL-15-treated 5637 cells. In addition, the cell-cycle inhibitor p21WAF1 was induced by the addition of IL-15. Finally, the siRNA-mediated knockdown of p21WAF1 attenuated the IL-15-induced stimulation of migration, invasion, ERK1/2 activation, MMP-9 expression, and NF-κB binding activation in 5637 cells. Our results suggest that p21WAF1 regulated NF-κB-mediated MMP-9 expression via the activation of ERK1/2, which resulted in the migration and invasion of 5637 cells treated with IL-15. These unexpected results suggest a potential role for IL-15 with respect to the progression of bladder cancer.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Interleucina-15/inmunología , Metaloproteinasa 9 de la Matriz/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias de la Vejiga Urinaria/inmunología , Butadienos/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Metaloproteinasa 9 de la Matriz/genética , FN-kappa B/metabolismo , Invasividad Neoplásica , Nitrilos/farmacología , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteína p53 Supresora de Tumor/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Interleukin-7 (IL-7) plays a pivotal role in the development and survival of lymphocytes, but its role in cancer cell responses remains unexplained. In this study, IL-7 treatment resulted in a significant induction in the wound-healing migration and Matrigel invasion of the 5637 bladder cancer cells, but it did not result in cell proliferation. In addition, IL-7 treatment strongly induced MMP-9 expression, and increased the binding activation of NF-κB and AP-1 motifs, the important transcription factors that regulate MMP-9 expression. Moreover, the treatment of 5637 cells with IL-7 stimulated the phosphorylation of ERK1/2. U0126, an ERK1/2-specific inhibitor, blocked IL-7-induced cell migration and invasion, and also suppressed the expression of MMP-9 in the presence of IL-7. Inhibition of the ERK1/2 function consistently reversed the binding activity of NF-κB without altering AP-1 activation in IL-7-stimulated cells. Among the cell cycle regulators examined, only the expression of the cell cycle inhibitor p27KIP1 was induced by IL-7. Moreover, the inhibition of p27KIP1 by small interfering RNA (siRNA) abolished the migration, invasion and phosphorylation of ERK1/2, the expression of MMP-9, and the binding activity of the NF-κB motif in IL-7-stimulated 5637 cells. These results demonstrated that the cell cycle inhibitor p27KIP1 is involved in ERK1/2-mediated MMP-9 expression via activation of the NF-κB binding motif, which leads to the migration and invasion of bladder cancer cells induced by IL-7. These novel results could help explain the migration and invasion of bladder tumor cells.
Asunto(s)
Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Interleucina-7/farmacología , Metaloproteinasa 9 de la Matriz/biosíntesis , FN-kappa B/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/biosíntesis , Humanos , Invasividad Neoplásica/patología , Fosforilación/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño , Factor de Transcripción AP-1/biosíntesis , Neoplasias de la Vejiga Urinaria/patología , Cicatrización de Heridas/efectos de los fármacosRESUMEN
In this study, we investigated the effects of a selective urotensin II (UII) receptor antagonist, SB-657510, on the inflammatory response induced by UII in human umbilical vein endothelial cells (EA.hy926) and human monocytes (U937). UII induced inflammatory activation of endothelial cells through expression of proinflammatory cytokines (IL-1ß and IL-6), adhesion molecules (VCAM-1), and tissue factor (TF), which facilitates the adhesion of monocytes to EA.hy926 cells. Treatment with SB-657510 significantly inhibited UII-induced expression of IL-1ß, IL-6, and VCAM-1 in EA.hy926 cells. Further, SB-657510 dramatically blocked the UII-induced increase in adhesion between U937 and EA.hy926 cells. In addition, SB-657510 remarkably reduced UII-induced expression of TF in EA.hy926 cells. Taken together, our results demonstrate that the UII antagonist SB-657510 decreases the progression of inflammation induced by UII in endothelial cells.