SP600125 Research Articles

Rotenone Induces a Neuropathological Phenotype in Cholinergic-like Neurons Resembling Parkinson’s Disease Dementia (PDD)

Parkinson’s disease with dementia (PDD) is a neurological disorder that clinically and neuropathologically overlaps with Parkinson’s disease (PD) and Alzheimer’s disease (AD). Although it is assumed that alpha-synuclein (α\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mathrm{\\alpha }$$\\end{document}-Syn), amyloid beta (Aβ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upbeta$$\\end{document}), and the protein Tau might synergistically induce cholinergic neuronal degeneration, presently the pathological mechanism of PDD remains unclear. Therefore, it is essential to delve into the cellular and molecular aspects of this neurological entity to identify potential targets for prevention and treatment strategies. Cholinergic-like neurons (ChLNs) were exposed to rotenone (ROT, 10μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upmu$$\\end{document}M) for 24 h. ROT provokes loss of ΔΨm\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\Delta \\mathrm{\\Psi m}$$\\end{document}, generation of reactive oxygen species (ROS), phosphorylation of leucine-rich repeated kinase 2 (LRRK2 at Ser935) concomitantly with phosphorylation of α\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mathrm{\\alpha }$$\\end{document} -synuclein (α\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mathrm{\\alpha }$$\\end{document}-Syn, Ser129), induces accumulation of intracellular Aβ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upbeta$$\\end{document} (iAβ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upbeta$$\\end{document}), oxidized DJ-1 (Cys106), as well as phosphorylation of TAU (Ser202/Thr205), increases the phosphorylation of c-JUN (Ser63/Ser73), and increases expression of proapoptotic proteins TP53, PUMA, and cleaved caspase 3 (CC3) in ChLNs. These neuropathological features resemble those reproduced in presenilin 1 (PSEN1) E280A ChLNs. Interestingly, anti-oxidant and anti-amyloid cannabidiol (CBD), JNK inhibitor SP600125 (SP), TP53 inhibitor pifithrin-α\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mathrm{ \\alpha }$$\\end{document} (PFT), and LRRK2 kinase inhibitor PF-06447475 (PF475) significantly diminish ROT-induced oxidative stress (OS), proteinaceous, and cell death markers in ChLNs compared to naïve ChLNs. In conclusion, ROT induces p-α\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mathrm{ \\alpha }$$\\end{document} -Syn, iAβ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upbeta$$\\end{document}, p-Tau, and cell death in ChLNs, recapitulating the neuropathology findings in PDD. Our report provides an excellent in vitro model to test for potential therapeutic strategies against PDD. Our data suggest that ROT induces a neuropathologic phenotype in ChLNs similar to that caused by the mutation PSEN1 E280A.

Combination of Tramiprosate, Curcumin, and SP600125 Reduces the Neuropathological Phenotype in Familial Alzheimer Disease PSEN1 I416T Cholinergic-like Neurons.

Familial Alzheimer's disease (FAD) is a complex and multifactorial neurodegenerative disorder for which no curative therapies are yet available. Indeed, no single medication or intervention has proven fully effective thus far. Therefore, the combination of multitarget agents has been appealing as a potential therapeutic approach against FAD. Here, we investigated the potential of combining tramiprosate (TM), curcumin (CU), and the JNK inhibitor SP600125 (SP) as a treatment for FAD. The study analyzed the individual and combined effects of these two natural agents and this pharmacological inhibitor on the accumulation of intracellular amyloid beta iAβ; hyperphosphorylated protein TAU at Ser202/Thr205; mitochondrial membrane potential (ΔΨm); generation of reactive oxygen species (ROS); oxidized protein DJ-1; proapoptosis proteins p-c-JUN at Ser63/Ser73, TP53, and cleaved caspase 3 (CC3); and deficiency in acetylcholine (ACh)-induced transient Ca2+ influx response in cholinergic-like neurons (ChLNs) bearing the mutation I416T in presenilin 1 (PSEN1 I416T). We found that single doses of TM (50 μM), CU (10 μM), or SP (1 μM) were efficient at reducing some, but not all, pathological markers in PSEN 1 I416T ChLNs, whereas a combination of TM, CU, and SP at a high (50, 10, 1 μM) concentration was efficient in diminishing the iAβ, p-TAU Ser202/Thr205, DJ-1Cys106-SO3, and CC3 markers by -50%, -75%, -86%, and -100%, respectively, in PSEN1 I417T ChLNs. Although combinations at middle (10, 2, 0.2) and low (5, 1, 0.1) concentrations significantly diminished p-TAU Ser202/Thr205, DJ-1Cys106-SO3, and CC3 by -69% and -38%, -100% and -62%, -100% and -62%, respectively, these combinations did not alter the iAβ compared to untreated mutant ChLNs. Moreover, a combination of reagents at H concentration was able to restore the dysfunctional ACh-induced Ca2+ influx response in PSEN 1 I416T. Our data suggest that the use of multitarget agents in combination with anti-amyloid (TM, CU), antioxidant (e.g., CU), and antiapoptotic (TM, CU, SP) actions might be beneficial for reducing iAβ-induced ChLN damage in FAD.

Piceatannol selectively inhibited the JNK3 enzyme and augmented apoptosis through inhibition of Bcl-2/Cyt-c/caspase-dependent pathways in the oxygen-glucose deprived SHSY-5Y cell lines: In silico and invitro study.

JNK3, a neuronal kinase activated by stress, plays a role in stress-induced apoptosis, leading to neuronal cell death following cerebral ischemia. This study investigates the neuroprotective effects of piceatannol (PCT) in SHSY-5Y neuroblastoma cells after hypoxic injury and its interaction with JNK3. We analyzed the crystal coordinates, interaction energies, and amino acid interactions to determine PCT's selectivity for JNK3. The electrostatic potential was computed using density functional theory, while molecular dynamics assessed the stability and structural consistency of the JNK3-PCT complex. We used SP600125 (SP6), a JNK3 inhibitor, as a reference compound. Additionally, we performed cell-free JNK 1, 2, and 3 kinase assays to evaluate the isoform selectivity of PCT. Cytotoxicity and cell viability were determined by an MTT test. To assess apoptosis, we used acridine orange/ethidium bromide dual fluorescent labeling and ANNEXIN A5-FITC flow cytometry. Western blot was used to evaluate the attenuation of JNK3 and apoptotic proteins. In silico studies revealed a stronger binding affinity between PCT and JNK3 compared to JNK1 and JNK2, which was further supported by the invitro kinase assay. PCT-treated cells exhibited a decrease in Cyt-c and caspase-3 expression and an increase in Bcl-2 level, compared to hypoxic control (p < .001). PCT also demonstrated superior efficacy over SP6 in inhibiting JNK3 phosphorylation (p < .001). Furthermore, PCT significantly increased the expression of neuronal genes, including NgN1, neuroD2, and survivin (p < .001). In conclusion, PCT is a potential JNK3 inhibitor, since it inhibited phosphorylation and the Bcl-2/Cyt-C/caspase-3-dependent apoptotic pathway after ischemic/hypoxic insult.

Design and Synthesis of a Mitochondrial-Targeted JNK Inhibitor and Its Protective Effect on Parkinson's Disease Phenotypes.

C-Jun N-terminal kinase (JNK) is a key mediator involved in a variety of physiological processes. JNK activation is regulated in a complex manner by upstream kinases and phosphatases, and plays an important role in physiological processes such as the immune response and neuronal function. Therefore, JNK has become a therapeutic target for neurodegenerative diseases, ankylosing spondylitis, psoriasis, arthritis and other diseases. Inhibition of JNK activation in mitochondria holds great potential for Parkinson's disease (PD) therapy. However, no specific mitochondrial-targeted JNK inhibitor has been reported. We have developed a mitochondrial-targeted JNK inhibitor, P2, by linking a mitochondrial-specific cell-penetrating peptide to SP600125 (SP), a commercialized specific inhibitor of JNK. We found that P2 specifically inhibited mitochondrial JNK phosphorylation instead of nuclear JNK signaling. Further studies showed that P2 effectively rescued PD phenotypes both in vitro and in vivo, thus indicating that it is a potential therapeutic for PD.

LSR antibody promotes apoptosis and disrupts epithelial barriers via signal pathways in endometrial cancer

ABSTRACT Lipolysis-stimulated lipoprotein receptor (LSR), a lipid metabolism-related factor localized in tricellular tight junctions (tTJs), plays an important role in maintaining the epithelial barrier. LSR is highly expressed in well-differentiated endometrial endometrioid carcinoma (EEC), and its expression decreases during malignancy. Angubindin-1, a novel LSR ligand peptide, regulates tTJs without cytotoxicity, enhances paracellular permeability, and regulates epithelial barrier via c-Jun N-terminal kinase (JNK)/cofilin. In this study, we investigated the immune-modulatory roles of an anti-LSR antibody in the treatment of EEC in vitro compared to those of angubindin-1. We prepared an antibody against the extracellular N-terminal domain of human LSR (LSR-N-ab) and angubindin-1. EEC cell-line Sawano cells in 2D and 2.5D cultures were treated with 100 μg/ml LSR-N-ab or 2.5 μg/ml angubindin-1 with or without protein tyrosine kinase 2β inhibitor PF431396 (PF43) and JNK inhibitor SP600125 (SP60) at 10 μM. Treatment with LSR-N-ab and angubindin-1 decreased LSR at the membranes of tTJs and the activity of phosphorylated LSR and phosphorylated cofilin in 2D culture. Treatment with LSR-N-ab and angubindin-1 decreased the epithelial barrier measured as TEER values in 2D culture and enhanced the epithelial permeability of FD-4 in 2.5D culture. Treatment with LSR-N-ab, but not angubindin-1, induced apoptosis in 2D culture. Pretreatment with PF43 and SP60 prevented all the changes induced by treatment with LSR-N-ab and angubindin-1. Treatment with LSR-N-ab and angubindin-1 enhanced the cell metabolism measured as the mitochondrial respiration levels in 2D culture. LSR-N-ab and angubindin-1 may be useful for therapy of human EEC via enhanced apoptosis or drug absorption.

2,5-dimethyl celecoxib induces apoptosis and autophagy via activation of ROS/JNK axis in nasopharyngeal carcinoma cells

2,5-dimethyl celecoxib (DMC), a close derivative of celecoxib, has also been reported to have anticancer effects. However, the effects and underlying molecular mechanisms of DMC with respect to nasopharyngeal carcinoma are still largely unknown. In this study, we present that DMC has displayed anticancer potency in nasopharyngeal carcinoma in vitro and in vivo. Mechanistically, we found DMC induced apoptosis and autophagy for anticancer therapy against nasopharyngeal carcinoma. Furthermore, DMC-induced autophagy could remarkably attenuate after the treatment of reactive oxygen species (ROS) scavenger N-acetyl cysteine (NAC) and c-Jun N-terminal kinase (JNK) inhibitor SP600125 (SP). Taken together, these results suggested DMC induced apoptosis and autophagic death via activation of ROS/JNK axis in NPC cells, which providing us new insights into developing potential therapeutic agents for nasopharyngeal carcinoma patients.

Alpinia oxyphylla Miq extract reduces cerebral infarction by downregulating JNK-mediated TLR4/T3JAM- and ASK1-related inflammatory signaling in the acute phase of transient focal cerebral ischemia in rats

BackgroundPost-ischemic inflammation is a crucial component in stroke pathology in the early phase of cerebral ischemia–reperfusion (I/R) injury. Inflammation caused by microglia, astrocytes, and necrotic cells, produces pro-inflammatory mediators and exacerbates cerebral I/R injury. This study evaluated the effects of the Alpinia oxyphylla Miq [Yi Zhi Ren (YZR)] extract on cerebral infarction at 1 day after 90 min of transient middle cerebral artery occlusion (MCAo) and investigated the molecular mechanisms underlying the regulation of c-Jun N-terminal kinase (JNK)-mediated inflammatory cascades in the penumbral cortex. Rats were intraperitoneally injected with the YZR extract at the doses of 0.2 g/kg (YZR-0.2 g), 0.4 g/kg (YZR-0.4 g), or 0.8 g/kg (YZR-0.8 g) at MCAo onset.ResultsYZR-0.4 g and YZR-0.8 g treatments markedly reduced cerebral infarction, attenuated neurological deficits, and significantly downregulated the expression of phospho-apoptosis signal-regulating kinase 1 (p-ASK1)/ASK1, tumor necrosis factor receptor-associated factor 3 (TRAF3), TRAF3-interacting JNK-activating modulator (T3JAM), ionized calcium-binding adapter molecule 1 (Iba1), p-JNK/JNK, inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor-α, toll-like receptor 4 (TLR4), glial fibrillary acidic protein (GFAP), nuclear factor-kappa B (NF-κB), and interleukin-6 in the penumbral cortex at 1 day after reperfusion. SP600125 (SP), a selective JNK inhibitor, had the same effects. Furthermore, Iba1- and GFAP-positive cells were colocalized with TLR4, and colocalization of GFAP-positive cells was found with NF-κB in the nuclei.ConclusionYZR-0.4 g and YZR-0.8 g treatments exerted beneficial effects on cerebral ischemic injury by downregulating JNK-mediated signaling in the peri-infarct cortex. Moreover, the anti-infarction effects of YZR extract treatments were partially attributed to the downregulation of JNK-mediated TLR4/T3JAM- and ASK1-related inflammatory signaling pathways in the penumbral cortex at 1 day after reperfusion.

C-Jun N-Terminal Kinase as a Therapeutic Target in Experimental Autoimmune Encephalomyelitis.

c-Jun N-terminal kinase (JNK) is upregulated during multiple sclerosis relapses and at the peak of experimental autoimmune encephalomyelitis (EAE). We aim to investigate the effects of pharmacological pan-JNK inhibition on the course of myelin oligodendrocyte glycoprotein (MOG35-55) EAE disease using in vivo and in vitro experimental models. EAE was induced in female C57BL/6JRj wild type mice using MOG35-55. SP600125 (SP), a reversible adenosine triphosphate competitive pan-JNK inhibitor, was then given orally after disease onset. Positive correlation between SP plasma and brain concentration was observed. Nine, but not three, consecutive days of SP treatment led to a significant dose-dependent decrease of mean cumulative MOG35-55 EAE severity that was associated with increased mRNA expression of interferon gamma (INF-γ) and tumor necrosis factor alpha (TNF-α) in the spinal cord. On a histological level, reduced spinal cord immune cell-infiltration predominantly of CD3+ T cells as well as increased activity of Iba1+ cells were observed in treated animals. In addition, in vitro incubation of murine and human CD3+ T cells with SP resulted in reduced T cell apoptosis and proliferation. In conclusion, our study demonstrates that pharmacological pan-JNK inhibition might be a treatment strategy for autoimmune central nervous system demyelination.

Activation of PERK Contributes to Apoptosis and G2/M Arrest by Microtubule Disruptors in Human Colorectal Carcinoma Cells

Microtubule-targeting agents (MTAs) are widely used in cancer chemotherapy, but the therapeutic responses significantly vary among different tumor types. Protein kinase RNA-like endoplasmic reticular (ER) kinase (PERK) is an ER stress kinase, and the role of PERK in the anticancer effects of MTAs is still undefined. In the present study, taxol (TAX) and nocodazole (NOC) significantly induced apoptosis with increased expression of phosphorylated PERK (pPERK; Tyr980) in four human colon cancer cell lines, including HCT-15, COLO205, HT-20, and LOVO cells. Induction of G2/M arrest by TAX and NOC with increases in phosphorylated Cdc25C and cyclin B1 protein were observed in human colon cancer cells. Application of the c-Jun N-terminal kinase (JNK) inhibitors SP600125 (SP) and JNK inhibitor V (JNKI) significantly reduced TAX- and NOC-induced apoptosis and G2/M arrest of human colon cancer cells. Interestingly, TAX- and NOC-induced pPERK (Tyr980) protein expression was inhibited by adding the JNK inhibitors, SP and JNKI, and application of the PERK inhibitor GSK2606414 (GSK) significantly reduced apoptosis and G2/M arrest by TAX and NOC, with decreased pPERK (Tyr980) and pJNK, phosphorylated Cdc25C, and Cyc B1 protein expressions in human colon cancer cells. Decreased viability by TAX and NOC was inhibited by knockdown of PERK using PERK siRNA in COLO205 and HCT-15 cells. Disruption of the mitochondrial membrane potential and an increase in B-cell lymphoma-2 (Bcl-2) protein phosphorylation (pBcl-2; Ser70) by TAX and NOC were prevented by adding the PERK inhibitor GSK and JNK inhibitor SP and JNKI. A cross-activation of JNK and PERK by TAX and NOC leading to anti-CRC actions including apoptosis and G2/M arrest was first demonstrated herein.

Picroside II Protects SH-SY5Y Cells From Autophagy and Apoptosis Following Oxygen Glucose Deprivation/Reoxygen Injury by Inhibiting JNK Signal Pathway.

To explore the neuroprotective effect of picroside (Picr) II on C-Jun NH2-terminal kinase (JNK) signal pathway after oxygen glucose deprivation/reoxygen (OGD/R) in SH-SY5Y cells. In vitro, SH-SY5Y cells were used to establish the OGD/R model, which was divided into the control group, model group, Picr group, and SP600125 (SP) group. Cellular viability was measured by CCK8. Cytotoxicity was assessed with LDH assay kit. Ad-GFP-mRFP-LC3 was used to monitor autophagosome and autolysosome. Apoptoic cells were detected by Annexin V-FITC/PI apoptosis detection kit. The expressions of phospho-JNK and phospho-c-Jun were determined by western blot (WB) and immunofluorescence. The expressions of phospho-MKK4, phospho-Bcl-2, Bax, Beclin-1, and LC3 I/II were determined by WB. In the control group, only limited apoptosis and autophagy was observed, and the expression of associated proteins was very low. After OGD/R, the cellular viability of SH-SY5Y cells was reduced, whereas the cytotoxicity, apoptosis, and autophagy were increased, accompanied with an increase of phospho-MKK4, phospho-JNK, phospho-c-Jun, phospho-Bcl-2, LC3 II, Beclin-1, and Bax. During the reoxygen, treatment with Picr II or SP600125 could strengthen the cellular viability of SH-SY5Y cells, but repress the cytotoxicity, apoptosis, autophagy, and the expressions of associated protein. OGD/R could induce apoptosis and autophagy of SH-SY5Y cells by activating JNK signal pathway. Picr II could protect SH-SY5Y cells from autophagy and apoptosis following OGD/R by inhibiting JNK signal pathway. Anat Rec, 302:2245-2254, 2019. © 2019 American Association for Anatomy.

Nilotinib induction of melanogenesis via reactive oxygen species-dependent JNK activation in B16F0 mouse melanoma cells.

Nilotinib (AMN), a second-generation tyrosine kinase inhibitor, induces apoptosis in various cancer cells, and our recent study showed that AMN effectively reduced the viability of human ovarian cancer cells via mitochondrion-dependent apoptosis. The effect of AMN in the melanogenesis of melanoma cells is still unclear. In the present study, we found that the addition of AMN but not imatinib (STI) significantly increased the darkness of B16F0 melanoma cells, and the absorptive value increased with the concentration of AMN. A decrease in the viability of B16F0 cells by AMN was detected in a concentration-dependent manner, accompanied by increased DNA ladders, hypodiploid cells and cleavage of the caspase-3 protein. An in vitro tyrosinase (TYR) activity assay showed that increased TYR activity by AMN was detected in a concentration-dependent manner; however, induction of TYR activity by STI at a concentration of 40μmol/L was observed. Increased intracellular peroxide by AMN was detected in B16F0 cells, and application of the antioxidant, N-acetylcysteine (NAC), significantly reduced AMN-induced peroxide production which also reduced the darkness of B16F0 cells. Additionally, AMN induced c-Jun N-terminal kinase (JNK) protein phosphorylation in B16F0 cells, which was inhibited by the addition of NAC. AMN-induced melanogenesis of B16F0 cells was significantly inhibited by the addition of NAC and the JNK inhibitor, SP600125 (SP). Data of Western blotting showed that increased protein levels of melanogenesis-related enzymes of tyrosinase-related protein-1 (TRP1), TRP2 and TYR were observed in AMN-treated B16F0 cells which were inhibited by the addition of NAC and SP. Evidence is provided supporting AMN effectively inducing the melanogenesis of B16F0 melanoma cells via reactive oxygen species-dependent JNK activation.

Silencing of H4R inhibits the production of IL-1β through SAPK/JNK signaling in human mast cells

Context: Mast cell (MC) activation through H4R releases various inflammatory mediators which are associated with allergic asthma.Objectives: To investigate the siRNA-mediated gene silencing effect of H4R on human mast cells (HMCs) functions and the activation of stress-activated protein kinases (SAPK)/jun amino-terminal kinases (JNK) signaling pathways for the release of ineterleukin-1β (IL-1β) in HMCs.Materials and methods: H4R expression was analyzed by RT-PCR and western blotting in human mast cell line-1 (HMC-1) cells and H4RsiRNA transfected cells. The effect of H4RsiRNA and H4R-antagonist on H4R mediated MC functions such as intracellular Ca2+ release, degranulation, IL-6 and IL-1β release, and the activation SAPK/JNK signaling pathways were studied. HMC-1 cells were stimulated with 10 μM of histamine (His) and 4-methylhistamine (4-MH) and pretreated individually with H4R-antagonist JNJ7777120 (JNJ), histamine H1 receptor (H1R)-antagonist mepyramine, and signaling molecule inhibitors SP600125 (SP) and Bay117082.Results: We found that the HMC-1 cells expressed H4R and H4RsiRNA treatment down regulated the H4R expression in HMC-1 cells. Both His and 4-MH induced the intracellular Ca2+ release and degranulation whereas; H4R siRNA and JNJ inhibited the effect. Furthermore, the activation of H4R caused the phosphorylation of SAPK/JNK pathways. H4R gene silencing and pretreatment with SP and JNJ decreased His and 4-MH induced phosphorylation of SAPK/JNK. We found that the activation of H4R caused the release of IL-1β (124.22 pg/ml) and IL-6 (122.50 pg/ml) on HMC-1 cells. Whereas, SAPK/JNK inhibitor (68.36 pg/ml) inhibited the H4R mediated IL-1β release.Conclusions: Taken together, the silencing of H4R inhibited the H4R mediated MC functions and SAPK/JNK phosphorylation. Furthermore, the H4R activation utilized SAPK/JNK signaling pathway for IL-1β release in HMC-1 cells.

SYKT Alleviates Doxorubicin-Induced Cardiotoxicity via Modulating ROS-Mediated p53 and MAPK Signal Pathways.

Backgrounds. Doxorubicin (DOX) is an effective therapeutic drug for malignant tumors; however, its clinical applications were limited by its side effects, especially the cardiotoxicity caused by ROS-mediated p53 and MAPK signal pathways' activation-induced cell apoptosis. Sanyang Xuedai mixture (SYKT) has been reported as an antioxidant agent and attenuated DOX-induced cardiotoxicity by targeting ROS-mediated apoptosis, but the mechanisms are still not fully delineated. Objective. This study aimed at investigating whether SYKT alleviated DOX-induced cardiotoxicity by inhibiting ROS-mediated apoptosis and elucidating the role of ROS-mediated p53 and MAPK signal pathways' activation in this process. Materials and Methods. Identification, separation, and culture of mouse primary cardiomyocytes. Cells were treated with DOX (1 μM), SYKT (30 mg/mL), or SYKT coupled with DOX. The p53 inhibitor Pifithrin-α (PFT-α), p38/MAPK inhibitor SB203583 (SB), and JNK inhibitor SP600125 (SP) were used as positive control. Western blot was employed to detected p53 and p38 as well as JNK expressions and the activation and translocation of Bax and cytochrome C. Flow cytometer (FCM) was used to detect the mitochondrial membrane potential and cell apoptosis. Results. After separation and culture, 95% of cells showed positive cTnI expression, which indicated that mouse primary cardiomyocytes were successfully identified in our research. DOX activated p53 and MAPK signal pathways in a time-dependent manner, which were inactivated by being cotreated with SYKT, PFT-α, or SB, respectively. DOX significantly decreased Bax and increased cytochrome c expressions in the cytoplasm, whereas Bax was upregulated and cytochrome c was downregulated in the mitochondria, which were reversed by SYKT treatment. Besides, DOX reduced mitochondria membrane potential (MMP) in cardiomyocytes compared to the control group; SYKT recovered its MMP and attenuated DOX-induced cardiomyocyte injury. Of note, DOX increased the expression levels of cleaved caspase-3 as well as poly ADP-ribose polymerase (PARP) and promoted cell apoptosis, which were also reversed by SYKT treatment. Discussion and Conclusions. Our results indicated that SYKT alleviated DOX-induced cardiotoxicity by inhibiting p53 and MAPK signal pathways' activation-mediated apoptosis, and it might serve as a potential therapeutic agent for DOX-induced cardiotoxicity.

Multicellular spheroids from normal and neoplastic thyroid tissues as a suitable model to test the effects of multikinase inhibitors.

Multicellular three-dimensional (3D) spheroids represent an experimental model that is intermediate in its complexity between monolayer cultures and patients’ tumor. In the present study, we characterize multicellular spheroids from papillary (PTC) and follicular (FTC) thyroid cancers and from the corresponding normal tissues. We show that these 3D structures well recapitulate the features of the original tissues, in either the differentiated and “stem-like” components. As a second step, we were aimed to test the effects of a small multikinase inhibitor, SP600125 (SP), previously shown to efficiently induce cell death in undifferentiated thyroid cancer monolayer cultures. We demonstrate the potent effect of SP on cell growth and survival in our 3D multicellular cultures. SP exerts its main effects through direct and highly significant inhibition of the ROCK pathway, known to be involved in the regulation of cell migration and β-catenin turnover. Consistently, SP treatment resulted in a significant decrease in β-catenin levels with respect to basal conditions in tumor but not in normal spheroids, indicating that the effect is promisingly selective on tumor cells.In conclusion, we provide the morphological and molecular characterization of thyroid normal and tumor spheroids. In this 3D model we tested in vitro the effects of the multikinase inhibitor SP and further characterized its mechanism of action in both normal and tumor spheroids, thus making it an ideal candidate for developing new drugs against thyroid cancer.

Inhibition of c-Jun N-terminal kinase signaling suppresses skin flap apoptosis in a rat ischemia and/or reperfusion model

BackgroundThe goals of this study were to validate the role of c-Jun N-terminal kinase (JNK) activation in skin flap apoptosis in a rat model of abdomen skin ischemia and/or reperfusion (IR) and to compare the protective effect of SP600125 and hydrogen-rich saline in skin IR injury. MethodsMale Sprague–Dawley rats were divided into five groups: one sham surgery group and four surgery groups. Before undergoing 3 h of IR management, the surgery groups were treated with normal saline (IR), dimethyl sulfoxide, SP600125 (SP), and hydrogen-rich saline (H). On the third postoperative day, blood perfusion of the flap was measured using Laser Doppler flowmeters. Hematoxylin and eosin staining was used to observe morphologic changes. Early apoptosis was observed using TdT-mediated dUTP-X nick end-labeling staining. pASK-1, pJNK, Bcl-2, and Bax were examined by immunodetection. Caspase-3 activity was also measured 24 h after reperfusion. ResultsCompared to the IR group and the dimethyl sulfoxide group, the SP group and the H group had larger skin flap survival area, more blood perfusion and lower levels of caspase-3 activity. The SP and the H groups had high expression levels of Bcl-2 and low expression levels of pASK-1 and pJNK. Bax was significantly decreased in the SP group. In addition, cell apoptosis was decreased in both the sham surgery and the H groups. ConclusionsIR-induced JNK phosphorylation was reduced by SP600125, indicating that JNK mediates the apoptosis pathways in rat skin. In the SP and the H groups, the apoptotic factors measured showed similar expression levels, indicating that JNK inhibition during IR may be associated with H-mediated protection against skin IR apoptosis.

Protein Kinase RNA-Like Endoplasmic Reticulum Kinase-Mediated Bcl-2 Protein Phosphorylation Contributes to Evodiamine-Induced Apoptosis of Human Renal Cell Carcinoma Cells.

We investigated the anticancer mechanism of evodiamine (EVO) against the viability of human A498 renal cell carcinoma (RCC) cells in vitro and in vivo. The in vitro study showed that EVO decreased the viability of A498 cells with the occurrence of apoptotic characteristics such as hypodiploid cells, DNA ladders, chromatin-condensed cells, and cleaved caspase (Casp)-3/poly(ADP ribose) polymerase (PARP) proteins. Pharmacological studies using chemical inhibitors of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) indicated that phosphorylation of the c-Jun N-terminal kinase (JNK) protein participated in EVO-induced cell death of A498 cells, and application of the JNK inhibitor, SP600125 (SP), inhibited EVO-induced cleavage of the Casp-3/PARP proteins and chromatin condensation according to Giemsa staining. EVO disruption of the mitochondrial membrane potential (MMP) with increased protein levels of the phosphorylated Bcl-2 protein (p-Bcl-2) was prevented by JNK inhibitors in A498 cells. A structure-activity relationship study showed that a methyl group at position 14 in EVO was important for its apoptotic effects and increased p-Bcl-2 protein in A498 cells. Furthermore, significant increases in the phosphorylated endoplasmic reticular stress protein, protein kinase RNA-like endoplasmic reticulum kinase (p-PERK at Thr980), by EVO were detected in A498 cells, and the PERK inhibitor, GSK2606414, significantly suppressed EVO-induced apoptosis, p-JNK, p-PERK, and cleaved PARP proteins. The in vivo study showed that EVO significantly reduced RCC growth elicited by a subcutaneous injection of A498 cells, and an increased protein level of p-PERK was observed according to an immunohistochemical analysis. Apoptosis by EVO was also demonstrated in other RCC cells such as 786-O, ACHN, and Caki-1 cells. This is the first study to demonstrate the anti-RCC effect of EVO via apoptosis in vitro and in vivo, and activation of JNK and PERK to induce Bcl-2 protein phosphorylation, which led to disruption of the MMP.

Evodiamine from Evodia rutaecarpa induces apoptosis via activation of JNK and PERK in human ovarian cancer cells

BackgroundEvodiamine (EVO; 8,13,13b,14-tetrahydro-14-methylindolo[2′3′-3,4]pyrido[2,1-b]quinazolin-5-[7H]-one derived from the traditional herbal medicine Evodia rutaecarpa was reported to possess anticancer activity; however, the anticancer mechanism of EVO against the viability of human ovarian cancer cells is still unclear. PurposeA number of studies showed that chemotherapeutic benefits may result from targeting the endoplasmic reticular (ER) stress signaling pathway. The objective of the study is to investigate the mechanism by which ER stress protein PERK plays in EVO-induced apoptosis of human ovarian cancer cells. MethodsCell death analysis was performed by MTT assay, DNA fragmentation assay, and Giemsa staining. DiOC6 staining was used for mitochondrial membrane potential measurement. Protein levels were analyzed by Western blotting. Pharmacological studies using MAPK inhibitors and PERK inhibitor GSK2606414 were involved. ResultsThe viability of human ovarian cancer cells A2780, A2780CP, ES-2, and SKOV-3 was inhibited by EVO at various concentrations in accordance with increases in the percentage of apoptotic cells, DNA ladders, and cleavage of caspase 3 and poly(ADP ribose) polymerase (PARP) proteins. Decreased viability of cells was reversed by adding caspase inhibitors VAD and DEVD in SKOV-3 and A2780CP cells, and incubation of cells with JNK inhibitor SP600125 (SP) and JNKI, but not other MAPK and AKT inhibitors including PD98059, SB203580, significantly prevented the apoptosis elicited by EVO in human ovarian cancer cells. Furthermore, increased expression of phospho-eIF2α (peIF2α) and phospho-PERK (pPERK) proteins was detected in EVO-treated human ovarian cancer cells, and that was inhibited by adding JNK inhibitors SP600125 and JNKI. Application of a PERK inhibitor GSK2606414 showed a significant protection of human ovarian cancer cells A2780 and A2780CP from EVO-induced apoptosis. EVO disruption of mitochondrial membrane potential (MMP) was also inhibited by adding JNK or PERK inhibitors. The structure-activity relationship study indicated that the alkyl group at position 14 in EVO is important for apoptosis induction via activation of JNK and PERK in human ovarian cancer cells. ConclusionEvidence supporting EVO induction of apoptosis via activation of JNK and PERK to disrupt MMP in human ovarian cancer cells is provided, and the alkyl at position 14 is a critical substitution for the apoptotic actions of EVO.

SP600125 has a remarkable anticancer potential against undifferentiated thyroid cancer through selective action on ROCK and p53 pathways.

Thyroid cancer is the most common endocrine malignancy with increasing incidence worldwide.The majority of thyroid cancer cases are well differentiated with favorable outcome. However, undifferentiated thyroid cancers are one of the most lethal human malignancies because of their invasiveness, metastatization and refractoriness even to the most recently developed therapies.In this study we show for the first time a significant hyperactivation of ROCK/HDAC6 pathway in thyroid cancer tissues, and its negative correlation with p53 DNA binding ability.We demonstrate that a small compound, SP600125 (SP), is able to induce cell death selectively in undifferentiated thyroid cancer cell lines by specifically acting on the pathogenic pathways of cancer development. In detail, SP acts on the ROCK/HDAC6 pathway involved in dedifferentiation and invasiveness of undifferentiated human cancers, by restoring its physiological activity level. As main consequence, cancer cell migration is inhibited and, at the same time, cell death is induced through the mitotic catastrophe. Moreover, SP exerts a preferential action on the mutant p53 by increasing its DNA binding ability. In TP53-mutant cells that survive mitotic catastrophe this process results in p21 induction and eventually lead to premature senescence. In conclusion, SP has been proved to be able to simultaneously block cell replication and migration, the two main processes involved in cancer development and dissemination, making it an ideal candidate for developing new drugs against anaplastic thyroid cancer.

Withaferin A-caused production of intracellular reactive oxygen species modulates apoptosis via PI3K/Akt and JNKinase in rabbit articular chondrocytes.

Withaferin A (WFA) is known as a constituent of Ayurvedic medicinal plant, Withania somnifera, and has been used for thousands of years. Although WFA has been used for the treatment of osteoarthritis (OA) and has a wide range of biochemical and pharmacologic activities, there are no findings suggesting its properties on chondrocytes or cartilage. The aim of the present study is to investigate the effects of WFA on apoptosis with focus on generation of intracellular reactive oxygen species (ROS). Here we showed that WFA significantly increased the generation of intracellular ROS in a dose-dependent manner. We also determined that WFA markedly leads to apoptosis as evidenced by accumulation of p53 by Western blot analysis. N-Acetyl-L-Cystein (NAC), an antioxidant, prevented WFA-caused expression of p53 and inhibited apoptosis of chondrocytes. We also found that WFA causes the activation of PI3K/Akt and JNKinase. Inhibition of PI3K/Akt and JNKinase with LY294002 (LY)/triciribine (TB) or SP600125 (SP) in WFA-treated cells reduced accumulation of p53 and inhibited fragmented DNA. Our findings suggested that apoptosis caused by WFA-induced intracellular ROS generation is regulated through PI3K/Akt and JNKinase in rabbit articular chondrocytes.Graphical

Differential regulation of anti-inflammatory genes by p38 MAP kinase and MAP kinase kinase 6

BackgroundConventional p38α inhibitors have limited efficacy in rheumatoid arthritis, possibly because p38 blockade suppresses the counter-regulatory mechanisms that limit inflammation. In contrast, targeting the upstream MAP kinase kinases, MKK3 and MKK6, partially maintains p38-mediated anti-inflammatory responses in bone marrow-derived macrophages (BMDM). In this study, we explored the mechanisms that preserve anti-inflammatory gene expression by evaluating differential regulation of IL-10 and p38-dependent anti-inflammatory genes in MKK3−/−, MKK6−/−, and p38 inhibitor-treated wildtype cells.MethodsBMDM from wild type (WT), MKK3−/−, and MKK6−/− mice were pre-treated with p38 inhibitor SB203580 (SB), JNK inhibitor SP600125 (SP), and/or ERK inhibitor PD98059 (PD) and stimulated with LPS. Supernatant protein levels were measured by multiplex bead immunoassay. mRNA expression was determined by qPCR and protein expression by Western blot analysis. De novo IL-10 mRNA synthesis was quantified in cells treated with ethynyl-uridine and LPS followed by reverse transcription and qPCR. mRNA half-life was measured in LPS-treated cells that were then incubated with actinomycin D ± SB203580.ResultsPre-treatment of WT BMDM with p38 inhibitor significantly reduced IL-10 production in the three groups, while ERK and JNK inhibitors had minimal effects. IL-10 production was significantly decreased in MKK3−/− BMDM compared with either WT or MKK6−/− cells. IL-10 mRNA expression was modestly reduced in MKK3−/− BMDM but was preserved in MKK6−/− cells compared with WT. De novo IL-10 mRNA synthesis was inhibited in MKK3−/− and p38 inhibitor pre-treated cells, but not MKK6−/− cells compared with WT. IL-10 mRNA half-life was markedly reduced in p38 inhibitor-treated WT cells while MKK-deficiency had minimal effect. DUSP1 mRNA levels were preserved in MKK-deficient cells but not in p38 inhibitor-treated WT cells. Tristetraprolin mRNA and protein levels were reduced in p38 inhibitor-treated WT cells compared with MKK6−/− cells.ConclusionUnlike p38-inhibition, the absence of MKK6 mostly preserves IL-10 and TTP protein expression in BMDM. MKK6-deficiency also spares DUSP1 and IL-1RA, which are key negative regulators of the inflammatory response. Together, these data suggest that MKK6 is a potential therapeutic target in RA.