RESUMEN
Aortic dissection (AD) is a life-threatening condition with a high mortality rate and without effective pharmacological therapies. Our previous study illustrated that leukocyte immunoglobulin-like receptor B4 (LILRB4) knockdown promoted the contractile phenotypic switch and apoptosis of AD cells. This study aimed to further investigate the role of LILRB4 in animal models of AD and elucidate its underlying molecular mechanisms. Animal models of AD were established using 0.1% beta-aminopropionitrile and angiotensin II and an in vitro model was developed using platelet-derived growth factor BB (PDGF-BB). The effects of LILRB4 knockdown on histopathological changes, pyroptosis, phenotype transition, extracellular matrix (ECM), and Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) pathways were assessed using a series of in vivo and in vitro assays. The effects of the JAK2 inhibitor AG490 on AD cell function, phenotypic transition, and ECM were explored. LILRB4 was highly expressed in AD and its knockdown increased survival rate, reduced AD incidence, and alleviated histopathological changes in the AD mouse model. Furthermore, LILRB4 knockdown promoted contractile phenotype switch, stabilized the ECM, and inhibited pyroptosis. Mechanistically, LILRB4 knockdown inhibited the JAK2/STAT3 signaling pathway. JAK2 inhibitor AG490 inhibited cell viability and migration, enhanced apoptosis, induced G0/G1 cell cycle arrest, and suppressed S-phase progression in PDGF-BB-stimulated human aortic smooth muscle cells. LILRB4 knockdown suppresses AD development by inhibiting pyroptosis and the JAK2/STAT3 signaling pathway.
Asunto(s)
Disección Aórtica , Modelos Animales de Enfermedad , Janus Quinasa 2 , Piroptosis , Factor de Transcripción STAT3 , Transducción de Señal , Animales , Humanos , Masculino , Ratones , Disección Aórtica/metabolismo , Disección Aórtica/patología , Disección Aórtica/genética , Técnicas de Silenciamiento del Gen , Janus Quinasa 2/metabolismo , Janus Quinasa 2/genética , Ratones Endogámicos C57BL , Piroptosis/genética , Factor de Transcripción STAT3/metabolismo , Tirfostinos/farmacologíaRESUMEN
Previous studies have shown that scutellarin inhibits the excessive activation of microglia, reduces neuronal apoptosis, and exerts neuroprotective effects. However, whether scutellarin regulates activated microglia-mediated neuronal apoptosis and its mechanisms remains unclear. This study aimed to investigate whether scutellarin can attenuate PC12 cell apoptosis induced by activated microglia via the JAK2/STAT3 signalling pathway. Microglia were cultured in oxygen-glucose deprivation (OGD) medium, which acted as a conditioning medium (CM) to activate PC12 cells, to investigate the expression of apoptosis and JAK2/STAT3 signalling-related proteins. We observed that PC12 cells apoptosis in CM was significantly increased, the expression and fluorescence intensity of the pro-apoptotic protein Bax and apoptosis-related protein cleaved caspase-3 were increased, and expression of the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) was decreased. Phosphorylation levels and fluorescence intensity of the JAK2/STAT3 signalling pathway-related proteins JAK2 and STAT3 decreased. After treatment with scutellarin, PC12 cells apoptosis as well as cleaved caspase-3 and Bax protein expression and fluorescence intensity decreased. The expression and fluorescence intensity of Bcl-2, phosphorylated JAK2, and STAT3 increased. AG490, a specific inhibitor of the JAK2/STAT3 signalling pathway, was used. Our findings suggest that AG490 attenuates the effects of scutellarin. Our study revealed that scutellarin inhibited OGD-activated microglia-mediated PC12 cells apoptosis which was regulated via the JAK2/STAT3 signalling pathway.
Asunto(s)
Apigenina , Apoptosis , Glucuronatos , Janus Quinasa 2 , Microglía , Factor de Transcripción STAT3 , Transducción de Señal , Animales , Apigenina/farmacología , Factor de Transcripción STAT3/metabolismo , Janus Quinasa 2/metabolismo , Glucuronatos/farmacología , Células PC12 , Apoptosis/efectos de los fármacos , Microglía/efectos de los fármacos , Microglía/metabolismo , Transducción de Señal/efectos de los fármacos , Ratas , Ratones , Caspasa 3/metabolismo , Glucosa/metabolismo , Fármacos Neuroprotectores/farmacología , Fosforilación/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismo , Tirfostinos/farmacologíaRESUMEN
Multiple sclerosis (MS) is an autoimmune inflammatory condition affecting the central nervous system, and experimental autoimmune encephalomyelitis (EAE) animal models have been extensively used to study it. T-helper 17 cells, which produce interleukin-17(IL-17), play crucial roles in MS pathogenesis, and the JAK2/STAT3 pathway has an essential function in their differentiation from naive CD4 + T cells. This study investigated the effects of the JAK2/STAT3 pathway inhibitor AG490 on EAE in vivo and in vitro, as well as the underlying mechanisms. AG490 ameliorated EAE severity and attenuated its typical symptoms by downregulating proteins associated with the JAK2/STAT3 pathway. Furthermore, it decreased T-helper 17 cell differentiation from naive CD4 + T cells by inactivating STAT3. In addition, it conferred protective effects against EAE by restoring autophagy. These findings indicate the potential of AG490 as a candidate anti-MS therapeutic.
Asunto(s)
Autofagia , Encefalomielitis Autoinmune Experimental , Janus Quinasa 2 , Ratones Endogámicos C57BL , Factor de Transcripción STAT3 , Transducción de Señal , Células Th17 , Tirfostinos , Animales , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Factor de Transcripción STAT3/metabolismo , Tirfostinos/farmacología , Autofagia/efectos de los fármacos , Autofagia/fisiología , Janus Quinasa 2/metabolismo , Janus Quinasa 2/antagonistas & inhibidores , Células Th17/efectos de los fármacos , Células Th17/metabolismo , Femenino , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Diferenciación Celular/efectos de los fármacos , RatonesRESUMEN
Central poststroke pain (CPSP) is a type of central neuropathic pain whose mechanisms remain unknown. Recently, we showed that activated astrocytes and microglial cells are present in the spinal cord of CPSP model mice. Activated glial cells exacerbate cerebral ischemic pathology by increasing the expression of inflammatory factors. However, the involvement of spinal glial cells in CPSP remains unknown. We hypothesized that spinal glial cell-derived molecules cause hyperexcitability or promoted the development of CPSP. In this study, we identified glial cell-derived factors involved in the development of CPSP using a bilateral common carotid occlusion (BCAO)-induced CPSP mouse model. Male ddY mice were subjected to BCAO for 30 min. The von Frey test assessed mechanical hypersensitivity in the right hind paw of mice. BCAO mice showed hypersensitivity to mechanical stimuli and astrocyte activation in the spinal cord 3 days after treatment. DNA microarray analysis revealed a significant increase in lipocalin 2 (LCN2), is known as neutrophil gelatinase-associated lipocalin, in the superficial dorsal horns of BCAO-induced CPSP model mice. LCN2 colocalized with GFAP, an astrocyte marker. Spinal GFAP-positive cells in BCAO mice co-expressed signal transducer and activator of transcription 3 (STAT3). The increase in the fluorescence intensity of LCN2 and GFAP in BCAO mice was suppressed by intrathecal injection of AG490, an inhibitor of JAK2 and downstream STAT3 activation, or anti-LCN2 antibody. Our findings indicated that LCN2 in spinal astrocytes may be a key molecule and may be partly involved in the development of CPSP.
Asunto(s)
Astrocitos , Modelos Animales de Enfermedad , Lipocalina 2 , Médula Espinal , Accidente Cerebrovascular , Animales , Masculino , Lipocalina 2/metabolismo , Ratones , Médula Espinal/metabolismo , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/complicaciones , Astrocitos/metabolismo , Factor de Transcripción STAT3/metabolismo , Neuralgia/metabolismo , Neuralgia/etiología , Janus Quinasa 2/metabolismo , Tirfostinos/farmacología , Proteína Ácida Fibrilar de la Glía/metabolismoRESUMEN
In recent years, bladder carcinoma (BC) has shown an increasing incidence, with poor patient outcomes. In clinical practice, BC is still mainly treated by surgery combined with chemoradiotherapy. However, as chemotherapy resistance of tumor cells becomes more and more obvious, it is urgent to find more effective BC treatment regimes. With the increasing application and growing attention paid to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in various neoplastic diseases, EGFR-TKIs have been considered as a new treatment direction in the future. In this study, the research team used AG1478, an EGFR-TKI, to intervene with the BC cell line T24. It was found that the cell activity was statistically decreased, the apoptosis was enhanced, and the cells were dominantly arrested in the G0/G1 phase, confirming the future therapeutic potential of EGFR-TKIs in BC. Besides, the research team further observed that AG1478 also promoted pyroptosis in T24 cells, and its mechanism is related to the induction of mitochondrial oxidative stress damage. The findings lay a more reliable foundation for the future application of EGFR-TKIs in BC.
Asunto(s)
Apoptosis , Puntos de Control del Ciclo Celular , Receptores ErbB , Mitocondrias , Inhibidores de Proteínas Quinasas , Quinazolinas , Tirfostinos , Neoplasias de la Vejiga Urinaria , Humanos , Receptores ErbB/metabolismo , Receptores ErbB/antagonistas & inhibidores , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Tirfostinos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Piroptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacosRESUMEN
BACKGROUND: In the hypothalamic paraventricular nucleus (PVN) of spontaneously hypertensive rats (SHRs), the expression of the testis-specific protein, Y-encoded-like 2 (TSPYL2) and the phosphorylation level of Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) are higher comparing with the normotensive Wistar Kyoto rats (WKY). But how they are involved in hypertension remains unclear. TSPYL2 may interact with JAK2/STAT3 in PVN to sustain high blood pressure during hypertension. METHODS: Knockdown of TSPYL2 via adeno-associated virus (AAV) carrying shRNA was conducted through bilateral microinjection into the PVN of SHR and WKY rats. JAK2/STAT3 inhibition was achieved by intraperitoneally or PVN injection of AG490 into the SHRs. Blood pressure (BP), plasma norepinephrine (NE), PVN inflammatory response, and PVN oxidative stress were measured. RESULTS: TSPYL2 knock-down in the PVN of SHRs but not WKYs led to reduced BP and plasma NE, deactivation of JAK2/STAT3, decreased expression of pro-inflammatory cytokine IL-1ß, and increased expression of anti-inflammatory cytokine IL-10 in the PVN. Meanwhile, AG490 administrated in both ways reduced the BP in the SHRs and deactivated JAK2/STAT3 but failed to change the expression of TSPYL2 in PVN. AG490 also downregulated expression of IL-1ß and upregulated expression of IL-10. Both knockdown of TSPYL2 and inhibition of JAK2/STAT3 can reduce the oxidative stress in the PVN of SHRs. CONCLUSION: JAK2/STAT3 is regulated by TSPYL2 in the PVN of SHRs, and PVN TSPYL2/JAK2/STAT3 is essential for maintaining high BP in hypertensive rats, making it a potential therapeutic target for hypertension.
Asunto(s)
Presión Sanguínea , Hipertensión , Janus Quinasa 2 , Núcleo Hipotalámico Paraventricular , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Factor de Transcripción STAT3 , Transducción de Señal , Animales , Janus Quinasa 2/metabolismo , Factor de Transcripción STAT3/metabolismo , Núcleo Hipotalámico Paraventricular/metabolismo , Núcleo Hipotalámico Paraventricular/enzimología , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Hipertensión/metabolismo , Hipertensión/fisiopatología , Masculino , Modelos Animales de Enfermedad , Estrés Oxidativo/efectos de los fármacos , Norepinefrina/metabolismo , Ratas , Tirfostinos/farmacología , FosforilaciónRESUMEN
Parkinson's disease (PD) is a neurodegenerative disease characterized by dopaminergic neuron death and neuroinflammation. Emerging evidence points to the involvement of the transient receptor potential melastatin 2 (TRPM2) channel in neuron death and glial activation in several neurodegenerative diseases. However, the involvement of TRPM2 in PD and specifically its relation to the neuroinflammation aspect of the disease remains poorly understood. Here, we hypothesized that AG490, a TRPM2 inhibitor, can be used as a treatment in a mouse model of PD. Mice underwent stereotaxic surgery for 6-hydroxydopamine (6-OHDA) administration in the right striatum. Motor behavioral tests (apomorphine, cylinder, and rotarod) were performed on day 3 post-injection to confirm the PD model induction. AG490 was then daily injected i.p. between days 3 to 6 after surgery. On day 6, motor behavior was assessed again. Substantia nigra (SNc) and striatum (CPu) were collected for immunohistochemistry, immunoblotting, and RT-qPCR analysis on day 7. Our results revealed that AG490 post-treatment reduced motor behavior impairment and nigrostriatal neurodegeneration. In addition, the compound prevented TRPM2 upregulation and changes of the Akt/GSK-3ß/caspase-3 signaling pathway. The TRPM2 inhibition also avoids the glial morphology changes observed in the PD group. Remarkably, the morphometrical analysis revealed that the ameboid-shaped microglia, found in 6-OHDA-injected animals, were no longer present in the AG490-treated group. These results indicate that AG490 treatment can reduce dopaminergic neuronal death and suppress neuroinflammation in a PD mouse model. Inhibition of TRPM2 by AG490 could then represent a potential therapeutical strategy to be evaluated for PD treatment.
Asunto(s)
Ratones Endogámicos C57BL , Neuroglía , Canales Catiónicos TRPM , Tirfostinos , Animales , Canales Catiónicos TRPM/antagonistas & inhibidores , Canales Catiónicos TRPM/metabolismo , Ratones , Masculino , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuroglía/patología , Tirfostinos/farmacología , Tirfostinos/uso terapéutico , Progresión de la Enfermedad , Oxidopamina/toxicidad , Modelos Animales de Enfermedad , Degeneración Nerviosa/patología , Degeneración Nerviosa/tratamiento farmacológico , Trastornos Parkinsonianos/patología , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/prevención & control , Sustancia Negra/efectos de los fármacos , Sustancia Negra/patología , Sustancia Negra/metabolismo , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/tratamiento farmacológicoRESUMEN
The blood-brain barrier (BBB) is instrumental in clearing toxic metabolites from the brain, such as amyloid-ß (Aß) peptides, and in delivering essential nutrients to the brain, like insulin. In Alzheimer's disease (AD) brain, increased Aß levels are paralleled by decreased insulin levels, which are accompanied by insulin signaling deficits at the BBB. Thus, we investigated the impact of insulin-like growth factor and insulin receptor (IGF1R and IR) signaling on Aß and insulin trafficking at the BBB. Following intravenous infusion of an IGF1R/IR kinase inhibitor (AG1024) in wild-type mice, the BBB trafficking of 125I radiolabeled Aß peptides and insulin was assessed by dynamic SPECT/CT imaging. The brain efflux of [125I]iodo-Aß42 decreased upon AG1024 treatment. Additionally, the brain influx of [125I]iodoinsulin, [125I]iodo-Aß42, [125I]iodo-Aß40, and [125I]iodo-BSA (BBB integrity marker) was decreased, increased, unchanged, and unchanged, respectively, upon AG1024 treatment. Subsequent mechanistic studies were performed using an in vitro BBB cell model. The cell uptake of [125I]iodoinsulin, [125I]iodo-Aß42, and [125I]iodo-Aß40 was decreased, increased, and unchanged, respectively, upon AG1024 treatment. Further, AG1024 reduced the phosphorylation of insulin signaling kinases (Akt and Erk) and the membrane expression of Aß and insulin trafficking receptors (LRP-1 and IR-ß). These findings reveal that insulin signaling differentially regulates the BBB trafficking of Aß peptides and insulin. Moreover, deficits in IGF1R and IR signaling, as observed in the brains of type II diabetes and AD patients, are expected to increase Aß accumulation while decreasing insulin delivery to the brain, which has been linked to the progression of cognitive decline in AD.
Asunto(s)
Péptidos beta-Amiloides , Barrera Hematoencefálica , Insulina , Transducción de Señal , Animales , Masculino , Ratones , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Insulina/metabolismo , Radioisótopos de Yodo , Ratones Endogámicos C57BL , Fragmentos de Péptidos/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Tomografía Computarizada por Tomografía Computarizada de Emisión de Fotón Único/métodos , Tirfostinos/farmacologíaRESUMEN
BACKGROUND: Myocardial infarction (MI) is one of the most deadly diseases in the world. Hyperoside (Hyp) has been shown to have a protective effect on cardiovascular function through various signaling pathways, but whether it can protect myocardial infarction by regulating JAK2/STAT3 signaling pathway is unknown. AIM OF THE STUDY: To investigate whether Hyp could protect the heart against myocardial infarction injury in mice by modulating JAK2/STAT3 signaling pathway and its potential mechanism. METHODS: In vivo experiments, the myocardial infarction model was established by ligating the left anterior descending coronary artery (LAD) of male C57BL/6 mice permanently. The mice were divided into seven groups: sham group, MI group, MI+Hyp (9 mg/kg), MI+Hyp (18 mg/kg) group, MI+Hyp (36 mg/kg) group, MI+Captopril group (15 mg/kg) group and MI+Hyp (36 mg/kg)+AG490 (7.5 mg/kg) group. Each group of animals were given different concentrations of hyperoside, positive control drug or inhibitor of JAK2/STAT3 singaling. After 14 days of administration, the electrocardiogram (ECG), echocardiography and serum myocardial injury markers were examined; Slices of mouse myocardial tissue were assessed for histopathological changes by HE, Masson and Sirius Red staining. TTC and TUNEL staining were used to evaluate the myocardial infarction area and cardiomyocytes apoptosis respectively. The expression of JAK2/STAT3 signaling pathway, apoptosis and autophagy-related proteins were detected by western blot. In vitro experiments, rat H9c2 cardiomyocytes were deprived of oxygen and glucose (OGD) to stimulate myocardial ischemia. The experiment was divided into seven groups: Control group, OGD group, OGD+Hyp (20 µM) group, OGD+Hyp (40 µM) group, OGD+Hyp (80 µM), OGD+Captopril (10 µM) group and OGD+Hyp (80 µM)+AG490 (100 µM) group. Myocardial cell damage and redox index were measured 12 h after OGD treatment. ROS content in cardiomyocytes was detected by immunofluorescence. Cardiomyocytes apoptosis was detected by flow cytometry. The expressions of JAK2/STAT3 signaling pathway-related proteins, apoptosis and autophagy related proteins were detected by western blot. RESULTS: In vivo, hyperoside could ameolirate ECG abnormality, increase cardiac function, reduce myocardial infarction size and significantly reduce myocardial fibrosis level and oxidation level. The experimental results in vitro showed that Hyp could reduce the ROS content in cardiomyocytes, decrease the level of oxidative stress and counteract the apoptosis induced by OGD injury . Both in vivo and in vitro experiments showed that hyperoside could increase phosphorylated JAK2 and STAT3, indicating that hyperoside could play a cardioprotective role by activating JAK2/STAT3 signaling pathway. It was also shown that hyperoside could increase the autophagy level of cardiomyocytes in vivo and in vitro. However the cardiomyocyte-protective effect of Hyp was abolished in combination with JAK2/ STAT3 signaling pathway inhibitor AG490. These results indicated that the protective effect of Hyp on cardiomyocyte injury was at least partially achieved through the activation of the JAK2/STAT3 signaling pathway. CONCLUSION: Hyp can significantly improve cardiac function, ameliorate myocardial hypertrophy and myocardial remodeling in MI mice. The mechanism may be related to improving mitochondrial autophagy of cardiomyocytes to maintain the advantage of autophagy, and blocking apoptosis pathway through phagocytosis, thus suppressing apoptosis level of cardiomyocytes. These effects of Hyp are achieved, at least in part, by activating the JAK2/STAT3 signaling pathway.
Asunto(s)
Janus Quinasa 2 , Ratones Endogámicos C57BL , Infarto del Miocardio , Miocitos Cardíacos , Quercetina , Quercetina/análogos & derivados , Factor de Transcripción STAT3 , Transducción de Señal , Animales , Factor de Transcripción STAT3/metabolismo , Janus Quinasa 2/metabolismo , Infarto del Miocardio/tratamiento farmacológico , Masculino , Miocitos Cardíacos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Quercetina/farmacología , Ratones , Apoptosis/efectos de los fármacos , Modelos Animales de Enfermedad , Ratas , Tirfostinos/farmacología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Because of a reduced sensitivity of BRAF-mutant colorectal cancers to BRAF inhibitor treatment when compared with BRAF-mutant melanoma, it is essential to develop efficient drugs to cope with this disease. The new 2-(4-bromophenyl)-3-arylacrylonitrile compound Briva was prepared in one step from commercially available starting compounds. Briva and two known thiophene analogs (Thio-Iva and Thio-Dam) were tested for their cytotoxic activity against various tumor cell lines including colorectal and breast cancer cells. The antitumor activities of the test compounds were assessed in vitro via the MTT assay, DAPI staining of nuclei, RT-PCR and immunoblotting, wound healing, clonogenic assay, collagen I adhesion assay, and kinase inhibition assays. A selective activity of Briva was observed against BRAFV600E-mutant HT-29 and COLO-201 colorectal carcinoma (CRC) cells. Briva caused inhibition of HT-29 clonogenic tumor growth and was found to induce cytotoxicity by activating the intrinsic apoptosis pathway. In addition, Briva reduced HT-29 cell adhesion and migration. Kinase inhibition experiments revealed that Briva inhibits VEGFR2. Thus, Briva can be considered as a promising antitumor compound against BRAFV600E-mutant colon carcinoma by targeting VEGFR2 tyrosine kinase and consequently reducing cell adhesion and metastasis formation.
Asunto(s)
Antineoplásicos , Neoplasias Colorrectales , Humanos , Proteínas Proto-Oncogénicas B-raf , Tirfostinos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Mutación , Ensayos Antitumor por Modelo de Xenoinjerto , Proliferación CelularRESUMEN
Chikungunya virus (CHIKV) is a globally public health threat. There are currently no medications available to treat CHIKV infection. High-throughput screening of 419 kinase inhibitors was performed based on the cytopathic effect method, and six kinase inhibitors with reduced cytopathic effects, including tyrphostin AG879 (AG879), tyrphostin 9 (A9), sorafenib, sorafenib tosylate, regorafenib, and TAK-632, were identified. The anti-CHIKV activities of two receptor tyrosine kinase inhibitors, AG879 and A9, that have not been previously reported, were selected for further evaluation. The results indicated that 50% cytotoxic concentration (CC50) of AG879 and A9 in Vero cells were greater than 30 µM and 6.50 µM, respectively and 50% effective concentration (EC50) were 0.84 µM and 0.36 µM, respectively. The time-of-addition and time-of-removal assays illustrated that both AG879 and A9 function in the middle stage of CHIKV life cycle. Further, AG879 and A9 do not affect viral attachment; however, they inhibit viral RNA replication, and exhibit antiviral activity against CHIKV Eastern/Central/South African and Asian strains, Ross River virus and Sindbis virus in vitro.
Asunto(s)
Antineoplásicos , Fiebre Chikungunya , Virus Chikungunya , Animales , Chlorocebus aethiops , Humanos , Virus Chikungunya/genética , Células Vero , Tirfostinos/farmacología , Tirfostinos/uso terapéutico , Línea Celular , Antivirales/farmacología , Antivirales/uso terapéutico , Replicación Viral , Inhibidores de Proteínas Quinasas/farmacología , Antineoplásicos/farmacologíaRESUMEN
Intestinal infection with C. perfringens is responsible for outbreaks of diarrhea in piglets. Janus kinase / signal transducer and activator of transcription (JAK/STAT) is a vital signaling pathway that regulates cellular activity and inflammatory response, closely correlated with multiple diseases development and advances. Currently, the potential effect of JAK/STAT on C. perfringens beta2 (CPB2) treatment on porcine intestinal epithelial (IPEC-J2) cells has not been explored. The expression of JAK/STAT genes or proteins in IPEC-J2 cells induced by CPB2 were observed by qRT-PCR and Western blot, and further used WP1066 to explore the effect of JAK2/STAT3 on mechanism employed by CPB2 on apoptosis, cytotoxicity, oxidative stress and inflammatory cytokines of IPEC-J2 cells. JAK2, JAK3, STAT1, STAT3, STAT5A and STAT6 were highly expressed in CPB2-induced IPEC-J2 cells, among which STAT3 had the highest expression. Moreover, apoptosis, cytotoxicity and oxidative stress were attenuated via blocking the activation of JAK2/STAT3 by using WP1066 in CPB2-treated IPEC-J2 cells. Furthermore, WP1066 significantly suppressed the secretion of interleukin (IL)-6, IL-1ß and TNF-α induced by CPB2 in IPEC-J2 cells.Our findings provide some insights into the functional roles of JAK2/STAT3 in piglets against to C. perfringens infection.
Asunto(s)
Infecciones por Clostridium , Clostridium perfringens , Transducción de Señal , Enfermedades de los Porcinos , Clostridium perfringens/fisiología , Quinasas Janus/metabolismo , Transducción de Señal/efectos de los fármacos , Línea Celular , Intestinos/citología , Intestinos/metabolismo , Animales , Porcinos , Perfilación de la Expresión Génica , Piridinas/farmacología , Tirfostinos/farmacología , Toxinas Bacterianas/toxicidad , Reacción en Cadena en Tiempo Real de la Polimerasa , Western Blotting , Infecciones por Clostridium/metabolismo , Infecciones por Clostridium/patología , Infecciones por Clostridium/veterinaria , Enfermedades de los Porcinos/metabolismo , Enfermedades de los Porcinos/patologíaRESUMEN
Enteroaggregative Escherichia coli (EAEC) has been identified as a new enteropathogen that causes acute and chronic diarrhea in children and travelers. One defining aspect of EAEC-pathogenesis is the induction of an inflammatory response in intestinal epithelium. In this study, we have found that EAEC-induced EGFR activation in human small intestinal and colonic epithelial was attenuated in the presence of a specific inhibitor of EGFR (Tyrphostin AG1478). Further, the aggregative stacked-brick type of adherence of this organism to both the cell lines and this pathogen-induced cytoskeletal rearrangement of these cells was also reduced in the presence of Tyrphostin AG1478. Moreover, EAEC-induced activation of downstream effectors (ERK-1/2, PI3K and Akt) of EGFR mediated cell signaling pathways were found to be suppressed in the presence of EGFR inhibitor. A decrease in IL-8 response in EAEC infected both the cell types were also noted in the presence of specific inhibitors of these downstream effectors, transcription factors and Tyrphostin AG1478. We propose that EAEC-induced activation of EGFR is quintessential for stacked-brick adherence of EAEC to human intestinal epithelial cells, their cytoskeletal rearrangements and stimulation of ERK-1/2 and PI3K/Akt mediated signal transduction pathways, resulting in the activation of NF-κB, AP-1, STAT-3 and finally IL-8 secretion by these cells.
Asunto(s)
Infecciones por Escherichia coli , Escherichia coli , Niño , Humanos , Adhesión Bacteriana , Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Escherichia coli/metabolismo , Infecciones por Escherichia coli/metabolismo , Interleucina-8/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Tirfostinos/farmacología , Tirfostinos/metabolismoRESUMEN
New N-alkylindole-substituted 2-(pyrid-3-yl)-acrylonitriles with putative kinase inhibitory activity and their (p-cymene)Ru(II) piano-stool complexes were prepared and tested for their antiproliferative efficacy in various cancer models. Some of the indole-based derivatives inhibited tumor cell proliferation at (sub-)micromolar concentrations with IC50 values below those of the clinically relevant multikinase inhibitors gefitinib and sorafenib, which served as positive controls. A focus was set on the investigation of drug mechanisms in HCT-116 p53-knockout colon cancer cells in order to evaluate the dependence of the test compounds on p53. Colony formation assays as well as experiments with tumor spheroids confirmed the excellent antineoplastic efficacy of the new derivatives. Their mode of action included an induction of apoptotic caspase-3/7 activity and ROS formation, as well as anti-angiogenic properties. Docking calculations with EGFR and VEGFR-2 identified the two 3-aryl-2-(pyrid-3-yl)acrylonitrile derivatives 2a and 2b as potential kinase inhibitors with a preferential activity against the VEGFR-2 tyrosine kinase. Forthcoming studies will further unveil the underlying mode of action of the promising new derivatives as well as their suitability as an urgently needed novel approach in cancer treatment.
Asunto(s)
Antineoplásicos , Inhibidores de Proteínas Quinasas , Tirfostinos , Humanos , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Proliferación Celular , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Indoles/síntesis química , Indoles/farmacología , Simulación del Acoplamiento Molecular , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Relación Estructura-Actividad , Proteína p53 Supresora de Tumor , Tirfostinos/síntesis química , Tirfostinos/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Células HCT116RESUMEN
Cadmium (Cd), a common heavy metal in the environment, is associated with cognitive impairment. In the present study, we carried out a preliminary inquiry to explore whether Cd causes neurotoxicity by regulating the JAK2/STAT3 signaling pathway and affecting the expression of klotho genes in vivo and in vitro, providing clues for the mechanism of Cd-induced cognitive dysfunction. The rat samples were injected with Cd chloride solution for 14 weeks, and the memory function of the rats was detected. Different concentrations of Cd and JAK2/STAT3 signaling pathway inhibitors were used to treat PC12 cells and thus detect the apoptosis rate. The protein expression levels of JAK2, p-JAK2, STAT3, p-STAT3, and klotho in rat and PC12 cell were detected by ELISA and Western blot, respectively. With the increase in exposure dose, the memory function of rats was severely impaired. The expression of p-JAK2 and p-STAT3 proteins was significantly up-regulated, whereas that of klotho was significantly down-regulated both in vivo and in vitro (p < 0.05). In comparison with the high-dose Cd exposure group, after adding tyrphostin AG490 (AG490), the apoptosis rate of PC12 cells increased, whereas the phosphorylation levels of JAK2 and STAT3 in the cells decreased significantly (p < 0.05). Cd exposure may cause neurotoxicity by regulating the JAK2/STAT3 signaling pathway and down-regulating klotho protein expression, leading to cognitive dysfunction.
Asunto(s)
Cadmio , Tirfostinos , Animales , Ratas , Apoptosis , Cadmio/toxicidad , Cadmio/metabolismo , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Transducción de Señal , Factor de Transcripción STAT3 , Tirfostinos/farmacología , Proteínas KlothoRESUMEN
Osteoarthritis (OA) is a complex chronic inflammatory disease characterized by articular degeneration and pain. Recent studies have identified interleukin 6 (IL-6) as a potential mediator leading to OA, but the therapeutic effects of inhibiting IL-6 signaling in intreating OA need to be further clarified. Here, we identified the intracellular signal transduction induced by recombinant IL-6 and focused on the impact of tyrphostin AG490 (a JAK2 inhibitor) on cartilage degeneration and OA pain. We found that IL-6 increased the inflammatory cytokines production and hypertrophic markers expression of primary mouse chondrocytes by activating JAK2/STAT3. Meanwhile, tyrphostin AG490 significantly attenuated articular degeneration and osteophyte formation in experimental mice with anterior cruciate ligament transection (ACLT) surgery. In vivo electrophysiological experiments showed that articular stimulation of IL-6 induced spinal hyperexcitability, which was prevented by coinjection of tyrphostin AG490. Specifically, compared with DMSO-treated ACLT mice, tyrphostin AG490 improved ambulate activity of mice and abolished the enhancement of serum bradykinin induced by IL-6. Together, we suggest that tyrphostin AG490 protected against progression of OA and improved OA prognosis by reducing cartilage degeneration and arthritis pain. Our findings provide further evidence for targeting IL-6 signaling in the treatment of OA.
Asunto(s)
Cartílago Articular , Osteoartritis , Animales , Tirfostinos/farmacología , Tirfostinos/uso terapéutico , Interleucina-6/metabolismo , Cartílago Articular/metabolismo , Osteoartritis/tratamiento farmacológico , Osteoartritis/metabolismo , Condrocitos , Dolor/tratamiento farmacológico , Dolor/metabolismo , Modelos Animales de EnfermedadRESUMEN
Combination therapies or multi-targeted drugs have been pointed out as an option to prevent the emergence of resistant clones, which could make long-term treatment more effective and translate into better clinical outcomes for cancer patients. The NT157 compound is a synthetic tyrphostin that leads to long-term inhibition of IGF1R/IRS1-2-, STAT3- and AXL-mediated signaling pathways. Given the importance of these signaling pathways for the development and progression of lung cancer, this disease becomes an interesting model for generating preclinical evidence on the cellular and molecular mechanisms underlying the antineoplastic activity of NT157. In lung cancer cells, exposure to NT157 decreased, in a dose-dependent manner, cell viability, clonogenicity, cell cycle progression and migration, and induced apoptosis (p < 0.05). In the molecular scenario, NT157 reduced expression of IRS1 and AXL and phosphorylation of p38 MAPK, AKT, and 4EBP1. Besides, NT157 decreased expression of oncogenes BCL2, CCND1, MYB, and MYC and increased genes related to cellular stress and apoptosis, JUN, BBC3, CDKN1A, CDKN1B, FOS, and EGR1 (p < 0.05), favoring a tumor-suppressive cell signaling network in the context of lung cancer. Of note, JNK was identified as a key kinase for NT157-induced IRS1 and IRS2 phosphorylation, revealing a novel axis involved in the mechanism of action of the drug. NT157 also presented potentiating effects on EGFR inhibitors in lung cancer cells. In conclusion, our preclinical findings highlight NT157 as a putative prototype of a multitarget drug that may contribute to the antineoplastic arsenal against lung cancer.
Asunto(s)
Antineoplásicos , Neoplasias Pulmonares , Pirogalol/análogos & derivados , Sulfonamidas/farmacología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis , Línea Celular Tumoral , Receptores ErbB/farmacología , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , MAP Quinasa Quinasa 4/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-bcl-2 , Proto-Oncogenes , Pirogalol/farmacología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Tirfostinos/farmacología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Schwannomatosis is a rare genetic disorder that predisposes individuals to development of multiple schwannomas mainly in spinal and peripheral nerves and to debilitating chronic pain often unrelated to any schwannoma. Pathogenic variants of two genes, SMARCB1 and LZTR1, are causal in familial cases. However, many schwannomatosis patients lack mutations in these genes. Surgery is the standard treatment for schwannomas but leaves patients with increasing neurological deficits. Pain management is a daily struggle controlled by the use of multiple analgesic and anti-inflammatory drugs. There is a need for both nonsurgical treatment to manage tumor growth and nonaddictive, nonsedative pain control. Because standard clinical trials are exceedingly difficult for patients with rare disorders, precision medicine approaches offer the possibility of bespoke therapeutic regimens to control tumor growth. As a proof of principle, we obtained a bio-specimen of paraspinal schwannoma from a schwannomatosis patient with a germline point mutation in the SMARCB1/INI gene. We created an hTERT immortalized cell line and tested the ability of targeted small molecules with efficacy in neurofibromatosis type 2-related schwannomas to reduce cell viability and induce cell death. We identified WP1066, a STAT3 inhibitor, currently in phase 2 clinical trials for pediatric and adult brain tumors as a lead compound. It reduced cell viability and STAT-3 phosphorylation and induced expression of markers for both necroptosis and caspase-dependent cell death. The results demonstrate feasibility in creating patient-derived cell lines for use in precision medicine studies.
Asunto(s)
Neurilemoma , Neurofibromatosis , Piridinas , Neoplasias Cutáneas , Tirfostinos , Adulto , Muerte Celular , Línea Celular Tumoral , Niño , Humanos , Neurilemoma/genética , Neurilemoma/patología , Neurofibromatosis/genética , Neurofibromatosis/patología , Piridinas/farmacología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Factores de Transcripción/genética , Tirfostinos/farmacologíaRESUMEN
Current options for preventing or treating influenza are still limited, and new treatments for influenza viral infection are urgently needed. In the present study, we serendipitously found that a small-molecule inhibitor (AG1478), previously used for epidermal growth factor receptor (EGFR) inhibition, demonstrated a potent activity against influenza both in vitro and in vivo. Surprisingly, the antiviral effect of AG1478 was not mediated by its EGFR inhibitory activity, as influenza virus was insensitive to EGFR blockade by other EGFR inhibitors or by siRNA knockdown of EGFR. Its antiviral activity was also interferon independent as demonstrated by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) knockout approach. Instead, AG1478 was found to target the Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1)-ADP-ribosylation factor 1 (ARF1) system by reversibly inhibiting GBF1 activity and disrupting its Golgi-cytoplasmic trafficking. Compared to known GBF1 inhibitors, AG1478 demonstrated lower cellular toxicity and better preservation of Golgi structure. Furthermore, GBF1 was found to interact with a specific set of viral proteins including M1, NP, and PA. Additionally, the alternation of GBF1 distribution induced by AG1478 treatment disrupted these interactions. Because targeting host factors, instead of the viral component, imposes a higher barrier for developing resistance, GBF1 modulation may be an effective approach to treat influenza infection.
Asunto(s)
Receptores ErbB , Factores de Intercambio de Guanina Nucleótido , Gripe Humana , Quinazolinas , Tirfostinos , Antivirales/farmacología , Receptores ErbB/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Gripe Humana/tratamiento farmacológico , Gripe Humana/metabolismo , Quinazolinas/farmacología , Transducción de Señal/efectos de los fármacos , Tirfostinos/farmacologíaRESUMEN
AIMS: Small molecule compound tyrphostin A9 (A9), an inhibitor of platelet-derived growth factor (PDGF) receptor, was previously reported by our group to stimulate extracellular signal-regulated kinase 1 (ERK1) and 2 (ERK2) in neuronal cells in a PDGF receptor-irrelevant manner. The study aimed to investigate whether A9 could protect axons in experimental autoimmune encephalomyelitis through activation of ERKs. MAIN METHODS: A9 treatment on the protection on neurite outgrowth in SH-SY5Y neuroblastoma cells and primary substantia nigra neuron cultures from the neurotoxin MPP+ were analyzed. Then, clinical symptoms as well as ERK1/2 activation, axonal protection induction, and the abundance increases of the regeneration biomarker GAP-43 in the CNS in the relapsing-remitting experimental autoimmune encephalomyelitis (EAE) model were verified. KEY FINDINGS: A9 treatment could stimulate neurite outgrowth in SH-SY5Y neuroblastoma cells and protect primary substantia nigra neuron cultures from the neurotoxin MPP+. In the relapsing-remitting EAE model, oral administration of A9 successfully ameliorated clinical symptoms, activated ERK1/2, induced axonal protection, and increased the abundance of the regeneration biomarker GAP-43 in the CNS. Interestingly, gene deficiency of ERK1 or ERK2 disrupted the beneficial effects of A9 in MOG-35-55-induced EAE. SIGNIFICANCE: These results demonstrated that small molecule compounds that stimulate persistent ERK activation in vitro and in vivo may be useful in protective or restorative treatment for neurodegenerative diseases.