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Objective To investigate the effect of SP600125 on the proliferation, cell cycle, apoptosis and invasion of human cervical cancer HeLa cells. Methods CCK-8 method was used to detect the proliferation of HeLa cells treated with different concentrations of SP600125 at different time points. The 20 μmol/L of SP600125 was determined for subsequent experiments. Cell proliferation ability was detected using plate clone formation assay; nuclear morphology was observed by DAPI staining; cell cycle and apoptosis were measured by flow cytometry; cell migration and invasion were detected by cell scratch and Transwell methods; the mRNA and protein levels of p53, Mad2L1 and CDC20 were measured by qRT-PCR and Western blot after SP600125 treatment at different time points. Results Compared with control group (0.1%DMSO), cells proliferative activity were reduced by 10, 20, 30, 40 and 50 μmol/L SP600125 treatment for 24h. Compared with control group, the rate of apoptosis was significantly increased in SP600125 treatment groups, and the cell proportion in G2/M phase increased (P < 0.001), while the cell proportion in G0/G1 phases cells was reduced after SP600125 treatment for 24h and 48h (P < 0.001), and the clonal number, migration and invasion ability of HeLa cells also decreased significantly (P < 0.001). qRT-PCR and Western blot results showed a significant decrease in Mad2L1 mRNA and protein expression (P < 0.05) and a significant increase in p53 and CDC20 mRNA and protein expression (P < 0.01). Conclusion SP600125 can induce cell cycle arrest of cervical cancer HeLa cells in G2/M phase by upregulating p53 and CDC20 and downregulating Mad2L1 expression, and promote cell apoptosis to inhibit cell proliferation, migration and invasion.
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Aim To investigate the effect of resveratrol (RSV)on apoptosis of PC 12 cells induced by advanced glycation end products(AGEs) and its possible molecular mechanism. Methods MTS assay was used to detect the effects of AGEs (0, 50, 100, 200, 400 g • L"1) and RSV (0, 5, 10, 20, 40 jxmol • L"1) on cell viability. PC12 cells were treated with AGEs (400 g • L-1) and RSV (10 |xmol • L"1). TUNEL staining was used to detect apoptosis; flow cytometry was used to detect apoptosis and reactive oxygen species (ROS) ; Western blot was used to detect protein expression of p-JNK, JNK, PUMA, Bcl-2, Bax and Caspase-3. Apoptosis was observed by flow cytometry after pretreat- ment with JNK specific phosphorylation inhibitor (sp600125). Results Compared with normal control group, the cell viability of AGEs group decreased, the apoptotic rate and ROS levels increased, the expressions of p-JNK, PUMA, Bax and Caspase-3 protein in-creased, the expression of Bcl-2 protein decreased; Compared with AGEs group, the cell viability of the AGEs + RSV group increased, the levels of apoptotic rate and ROS were reduced, the expressions of p-JNK, PUMA, Bax and caspase-3 protein decreased, the expression of Bcl-2 protein increased. Sp600125 could partially reverse the effect of AGEs on PC)2 cell apopto-sis. Conclusions RSV can significantly inhibit the apoptosis of PC 12 cells induced by AGEs, which may be related to the activation of ROS-JNK pathway.
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ABSTRACT Objective: To explore the application methods of mitogen-activated protein kinase signal pathway inhibitors SP600125 and SB203580 in long-term in vivo experiments. Methods: A total of 55 healthy New Zealand rabbits were randomly divided into blank control group, model control group, SP low dose group, SP high dose group, SP blank group, SB low dose group, SB high dose group, SB blank group, dimethyl sulfoxide (DMSO) control group, DMSO blank group, and positive control group. Since the first day of the experiment, each group was administered the corresponding treatment for four weeks continuously. Then, the myocardial c-Jun N-terminal kinase (JNK) and the total protein of p38, protein phosphorylation and its gene expression levels were detected. Results: After intravenous treatment with adriamycin, the myocardial phosphorylate-JNK (p-JNK) and phosphorylate-p38 (p-p38) levels in all groups were increased to varying degrees, of which the model control group increased the most significantly (p < 0.05). Compared with the model control group, the myocardial p-JNK and p-p38 increased more slowly in the SP low dose group, SP high dose group, SB low dose group, SB high dose group and positive control group (p < 0.05), of which the increase in the SP high dose group and the SB high dose group was the slowest (p < 0.05). After four weeks, the total protein and messenger ribonucleic acid of the myocardial JNK and p38 in all groups had no statistically significant difference (p > 0.05). Conclusion: The continuous intravenous injection of SP600125 and SB203580 for four weeks significantly reduced the protein phosphorylation levels of JNK and p38, which provides a practical avenue for the long-term study in vivo.
RESUMEN Objetivo: Explorar los métodos de aplicación de los inhibidores SP600125 y SB203580 de la vía de señalización de la proteína quinasa activada por mitógeno en experimentos in vivo a largo plazo. Métodos: Un total de 55 conejos sanos de Nueva Zelandia fueron divididos aleatoriamente en los grupos siguientes: grupo de control en blanco, grupo de control modelo, grupo de dosis baja SP, grupo de dosis alta SP, grupo en blanco SP, grupo de dosis baja SB, grupo de dosis alta SB, grupo en blanco SB, grupo de control dimetilsulfóxido (DMSO), grupo en blanco DMSO, y grupo de control positivo. Desde el primer día del experimento, a cada grupo se le administró el tratamiento correspondiente por cuatro semanas continuas. Entonces, se detectaron la quinasa c-Jun N-terminal (JNK) miocárdica y la proteína p38 total, así como la fosforilación proteica y sus niveles de expresión génica. Resultados: Después del tratamiento intravenoso con adriamicina, los niveles de fosfo-JNK (p-JNK) y fosfo-p38 (p-p38) del miocardio aumentaron en todos los grupos en diversos grados, siendo el aumento del grupo de control modelo el más significativo (p < 0.05). En comparación con el grupo de control modelo, p-JNK y p-p38 miocárdicos aumentaron más lentamente en el grupo de dosis baja SP, el grupo de dosis alta SP, el grupo de dosis baja SB, el grupo de dosis alta SB, y el grupo de control positivo (p < 0.05). De estos, el aumento en el grupo de dosis alta SP y el grupo de dosis alta SB fue el más lento (p < 0.05). Después de cuatro semanas, la proteína total y el ácido ribonucleico mensajero de JNK y p38 miocárdicos en todos los grupos, no tuvieron diferencias significativas (p > 0.05). Conclusión: La inyección intravenosa continua de SP600125 y SB203580 durante cuatro semanas redujo significativamente los niveles de fosforilación proteica de JNK y p38, lo que proporciona una vía práctica para el estudio a largo plazo in vivo.
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Humans , Male , Rabbits , Doxorubicin/pharmacology , Mitogen-Activated Protein Kinases/drug effects , Protein Kinase Inhibitors/pharmacology , Phosphorylation/drug effects , Time Factors , Signal Transduction/drug effects , Random Allocation , Gene ExpressionABSTRACT
Objective To elucidate the effects of SP600125 at different concentrations on the proliferation and osteo-differentiation of human adipose-derived stem cells (hASCs).Methods The hASCs harvested were cocuhured with SP600125 at concentrations of 0 μmol/L,1 μmol/L,5 μmol/L and 10 μmol/L in growth medium (OM group) and in osteogenesis medium (OM group),respectively.The DNA quantitative assay was carried out to evaluate proliferation of the hASCs;flow cytometry was used to determine the effect of SP600125 on the cell cycles of hASCs;Alkaline phosphatase level (ALP) and calcium deposition tests were conducted to observe the effects of SP600125 at different concentrations on osteogenic differentiation of the hASCs.Results The proliferation of hASCs was inhibited by 42.1％ when the cells were cocultured with SP600125 at the concentration of 10 μmol/L;the suppression decreased with decreased concentration of SP600125.The hASCs of phase G0/G1 in GM cocultured with SP600125 at the concentration of 10 μmol/L were more than those in GM cocultured with dimethylsulfoxide at the same concentration.ALP test revealed that after 10 days of culture in vitro the staining was more and more weakened and scattered and the ALP activity was more and more decreased with the increased concentration of SP600125.The extracellular calcium deposition of hASCs after 14 days of culture in vitro showed that the size and number of calcium nodules decreased with the increased concentration of SP600125.Conclusion SP600125 can suppress the proliferation and osteogenic differentiation of hASCs in vitro.
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In patients with advanced cancer, cancer-induced bone pain (CIBP) is a severe and common problem that is difficult to manage and explain. As c-Jun N-terminal kinase (JNK) and chemokine (C-X-C motif) ligand 1 (CXCL1) have been shown to participate in several chronic pain processes, we investigated the role of JNK and CXCL1 in CIBP and the relationship between them. A rat bone cancer pain model was established by intramedullary injection of Walker 256 rat gland mammary carcinoma cells into the left tibia of Sprague-Dawley rats. As a result, intramedullary injection of Walker 256 carcinoma cells induced significant bone destruction and persistent pain. Both phosphorylated JNK1 (pJNK1) and pJNK2 showed time-dependent increases in the ipsilateral spinal cord from day 7 to day 18 after tumor injection. Inhibition of JNK activation by intrathecal administration of SP600125, a selective pJNK inhibitor, attenuated mechanical allodynia and heat hyperalgesia caused by tumor inoculation. Tumor cell inoculation also induced robust CXCL1 upregulation in the ipsilateral spinal cord on day 18 after tumor injection. Inhibition of CXCL1 by intrathecal administration of CXCL1 neutralizing antibody showed a stable analgesic effect. Intrathecal administration of SP600125 reduced CXCL1 increase in the spinal cord, whereas inhibition of CXCL1 in the spinal cord showed no influence on JNK activation. Taken together, these results suggested that JNK activation in spinal cord contributed to the maintenance of CIBP, which may act through modulation of CXCL1. Inhibition of the pJNK/CXCL1 pathway may provide a new choice for treatment of CIBP.
Subject(s)
Animals , Female , Rats , Antibodies, Neutralizing , Allergy and Immunology , Therapeutic Uses , Bone Neoplasms , Metabolism , Cancer Pain , Drug Therapy , Metabolism , Cell Line, Tumor , Chemokine CXCL1 , Allergy and Immunology , Metabolism , JNK Mitogen-Activated Protein Kinases , Metabolism , Protein Kinase Inhibitors , Pharmacology , Therapeutic Uses , Rats, Sprague-Dawley , Spinal Cord , MetabolismABSTRACT
Objective To explore the protective effect and probable mechanism of JNK inhibitor SP600125 on hippocampal neurons in rats with status epilepsy following lithium?pilocarpine. Methods 48 Wistar rats,in accordance with the random number table,were divided into control,status epilepticus ( SE) and JNK in?hibitor SP600125 group ( SP ) . HE staining and fluorescent TUNEL method were used to observe pathological changes and neuronal apoptosis in the hippocampal area of rats in each group. Western blot was applied to detect the phosphorylation expression of JNK and its downstream effector molecule c?JUN in hippocampal tissues of rats in each group. Results Compared with control group,neuronal loss and apoptosis in CA3 area of hippocampus in SE group were significant (percentage of TUNEL positive cells (26.34±3.04)%, P<0.05). The mortality of rats was significantly decreased and neuronal loss and apoptosis were obviously reduced in SP group than in SE group ( mor?tality in SP and SE group :6.25%,37.5% respectively, P<0.05). Meanwhile,the expression levels of phospho?JNK and phospho?c?JUN were significantly increased in hippocampus of rats in SE group ( The relative OD values respectively 0.447±0.025,0.552±0.035, P<0.05 compared with Control group). After treated with SP600125 in SP group,the phosphorylation levels of JNK and c?JUN were obviously decreased ( The relative OD values respec?tively 0.211±0.016,0.237±0.028, P<0.05 compared with SE group). Conclusion JNK inhibitor SP600125 may play an important protective effect on neurons in the rat hippocampus after status epilepticus through inhibition of JNK and c?JUN phosphorylation.
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Objective: To investigate the relationship between the c-Jun N-terminal kinase (JNK) pathway and lung-resistance protein (LRP). Methods:A549 cells were treated with various concentrations of CDDP for 72 h. The LRP mRNA expression was then analyzed using reverse transcription PCR (RT-PCR). LRP, JNK, and P-JNK were analyzed by Western blotting. The A549 cells were then pretreated with SP600125 (2 μg/ml to 4 μg/ml ) for 1 h. Afterward, CDDP (16 μg/ml) was added into the culture for 72 h. A Cell Counting Kit-8 was used to investigate the sensitivity of CDDP to the A549 cells. Flow cytometry was used to detect the apoptosis rate. The LRP mRNA expression was analyzed using RT-PCR. LRP, JNK, and P-JNK were analyzed by Western blotting. Results:CDDP in-duced the mRNA and protein expression of both LRP and P-JNK in a dose-dependent manner. Pretreatment with SP600125 enhanced the sensitivity of CDDP to the A549 cells and increased the apoptosis rate. However, the LRP mRNA and LRP expression in the pre-treated cells was lower than that in the presence of CDDP alone. Conclusion:In A549 cells, CDDP induces the LRP expression via the JNK pathway. This result suggests that lung cancer therapy can be improved by the addition of CDDP to inhibit the JNK signaling path-way.
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Objective Taking CNE-2Z multicellular spheroids (MCSs) as the simulation of solid tumors, to investigate the role of SAPK/JNK signaling pathway in multicellular resistance to radiotherapy for human nasopharyngeal carcinoma (NPC). Methods Human NPC cell line CNE-2Z were cultured into multicellular spheroids by using liquid overlay technique, then divided into control MCSs, irradiated MCSs (average dose in one minute: 2 Gy), sp-600125(a specific inhibitor for SAPK/JNK signaling pathway)+irradiated MCSs, sp-600125+MCSs. Western blotting was employed to analyze the activity of SAPK/JNK signaling pathway in MCSs, and the expression of Caspase-3 protein before and after sp-600125 treatment; X-ray induced cell apoptotisis in MCSs before and after sp-600125 treatment was detected by TUNEL. Results The level of SAPK/JNK phosphorylation in MCSs was a dynamic course after radiation, and the phosphorylation peaked at 2 h after irradiation; The apoptotic rate of MCSs (P
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Objective To explore the effect of c-Jun N-terminal kinase(JNK)on the expression of interleukin-10(IL-10) in keratinocytes induced by ultraviolet A(UVA).Methods The HaCaT cells in cultured were either sham irradiated(control) or exposured to 2.4 J/cm2 UVA radiation.The cells were collected at 0-48 h after irradiation,and JNK levels in cells were detected with the immunofluorescence.HaCaT cells were treated with SP600125(a JNK inhibitor) before irradiation,then cells and suspended medium were collected at each time-point after irradiation,and the expression of IL-10 mRNA and protein were determined by RT-PCR and ELISA.Results Compared with control cells,irradiated cells had increased levels of phospho-JNK throughout the entire 48 h following irradiation(P