Programmed death of cardiomyocytes in cardiovascular disease and new therapeutic approaches.

Cardiovascular diseases (CVDs) have a complex pathogenesis and pose a major threat to human health. Cardiomyocytes have a low regenerative capacity, and their death is a key factor in the morbidity and mortality of many CVDs. Cardiomyocyte death can be regulated by specific signaling pathways known as programmed cell death (PCD), including apoptosis, necroptosis, autophagy, pyroptosis, and ferroptosis, etc. Abnormalities in PCD can lead to the development of a variety of cardiovascular diseases, and there are also molecular-level interconnections between different PCD pathways under the same cardiovascular disease model. Currently, the link between programmed cell death in cardiomyocytes and cardiovascular disease is not fully understood. This review describes the molecular mechanisms of programmed death and the impact of cardiomyocyte death on cardiovascular disease development. Emphasis is placed on a summary of drugs and potential therapeutic approaches that can be used to treat cardiovascular disease by targeting and blocking programmed cell death in cardiomyocytes.

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 iAß; 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 iAß, 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 iAß 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 iAß-induced ChLN damage in FAD.

Securinine inhibits the tumor growth of human bladder cancer cells by suppressing Wnt/ß-catenin signaling pathway and activating p38 and JNK signaling pathways.

Bladder cancer (BC) is the most common malignant tumor in urinary system. Although chemotherapy is one of the most important adjuvant treatments for BC, drug resistance, non-specific toxicity and severe side effects are the major obstacles to BC chemotherapy. Natural products have always been a leading resource of antitumor drug discovery, with the advantages of excellent effectiveness, low toxicity, multi-targeting potency and easy availability. In this study, we evaluated the potential anti-tumor effect of securinine (SEC), a natural alkaloid from Securinega suffruticosa, on BC cells in vitro and in vivo, and delineated the underlying mechanism. We found that SEC inhibited the proliferation, migration and invasion, induced the apoptosis of BC cells in vitro, and retarded the xenograft tumor growth of BC cell in vivo. Notably, SEC had a promising safety profile because it presented no or low toxicity on normal cells and mice. Mechanistically, SEC inactivated Wnt/ß-catenin signaling pathway while activated p38 and JNK signaling pathway. Moreover, ß-catenin overexpression, the p38 inhibitor SB203580 and the JNK inhibitor SP600125 both mitigated the inhibitory effect of SEC on BC cells. Furthermore, we demonstrated a synergistic inhibitory effect of SEC and gemcitabine (GEM) on BC cells in vitro and in vivo. Taken together, our findings suggest that SEC may exert anti-BC cell effect at least through the activation of p38 and JNK signaling pathways, and the inhibition of Wnt/ß-catenin signaling pathway. More meaningfully, the findings indicate that GEM-induced BC cell killing can be enhanced by combining with SEC.

Pharmacological Akt and JNK Kinase Inhibitors 10-DEBC and SP600125 Potentiate Anti-Glioblastoma Effect of Menadione and Ascorbic Acid Combination in Human U251 Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor in adults, characterized by a highly invasive nature and therapy resistance. Combination of menadione and ascorbic acid (AA+MD) exerts strong ROS-mediated anti-GBM activity in vitro. The objective of this study was to improve AA+MD anti-GBM potential by modulating the activity of Akt and c-Jun N-terminal kinase (JNK), molecules with an important role in GBM development. The effects of Akt and JNK modulation on AA+MD toxicity in U251 human glioblastoma cells were assessed by cell viability assays, flow cytometry, RNA interference and plasmid overexpression, and immunoblot analysis. The AA+MD induced severe oxidative stress, an early increase in Akt phosphorylation followed by its strong inhibition, persistent JNK activation, and U251 cell death. Small molecule Akt kinase inhibitor 10-DEBC enhanced, while pharmacological and genetic Akt activation decreased, AA+MD-induced toxicity. The U251 cell death potentiation by 10-DEBC correlated with an increase in the combination-induced autophagic flux and was abolished by genetic autophagy silencing. Additionally, pharmacological JNK inhibitor SP600125 augmented combination toxicity toward U251 cells, an effect linked with increased ROS accumulation. These results indicate that small Akt and JNK kinase inhibitors significantly enhance AA+MD anti-GBM effects by autophagy potentiation and amplifying deleterious ROS levels.

Lupenone attenuates thapsigargin-induced endoplasmic reticulum stress and apoptosis in pancreatic beta cells possibly through inhibition of protein tyrosine kinase 2 activity.

AIMS: Prolonged high levels of cytokines, glucose, or free fatty acids are associated with diabetes, elevation of cytosolic Ca2+ concentration ([Ca2+]C), and depletion of Ca2+ concentration in the endoplasmic reticulum (ER) of pancreatic beta cells. This Ca2+ imbalance induces ER stress and apoptosis. Lupenone, a lupan-type triterpenoid, is beneficial in diabetes; however, its mechanism of action is yet to be clarified. This study evaluated the protective mechanism of lupenone against thapsigargin-induced ER stress and apoptosis in pancreatic beta cells. MATERIALS AND METHODS: MIN6, INS-1, and native mouse islet cells were used. Western blot for protein expressions, measurement of [Ca2+]C, and in vivo glucose tolerance test were mainly performed. KEY FINDINGS: Thapsigargin increased the protein levels of cleaved caspase 3, cleaved PARP, and the phosphorylated form of JNK, ATF4, and CHOP. Thapsigargin increased the interaction between stromal interaction molecule1 (Stim1) and Orai1, enhancing store-operated calcium entry (SOCE). SOCE is further activated by protein tyrosine kinase 2 (Pyk2), which is Ca2+-dependent and phosphorylates the tyrosine residue at Y361 in Stim1. Lupenone inhibited thapsigargin-mediated Pyk2 activation, suppressed [Ca2+]C, ER stress, and apoptosis. Lupenone restored impaired glucose-stimulated insulin secretion effectuated by thapsigargin and glucose intolerance in a low-dose streptozotocin-induced diabetic mouse model. SIGNIFICANCE: These results suggested that lupenone attenuated thapsigargin-induced ER stress and apoptosis by inhibiting SOCE; this may be due to the hindrance of Pyk2-mediated Stim1 tyrosine phosphorylation. In beta cells that are inevitably exposed to frequent [Ca2+]C elevation, the attenuation of abnormally high SOCE would be beneficial for their survival.

JNK-JUN-NCOA4 axis contributes to chondrocyte ferroptosis and aggravates osteoarthritis via ferritinophagy.

Interruption of iron homeostasis is correlated with cell ferroptosis and degenerative diseases. Nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy has been reported as a vital mechanism to control cellular iron levels, but its impact on osteoarthritis (OA) pathology and the underline mechanism are unknown. Herein we aimed to investigate the role and regulatory mechanism of NCOA4 in chondrocyte ferroptosis and OA pathogenesis. We demonstrated that NCOA4 was highly expressed in cartilage of patients with OA, aged mice, post-traumatic OA mice, and inflammatory chondrocytes. Importantly, Ncoa4 knockdown inhibited IL-1ß-induced chondrocyte ferroptosis and extracellular matrix degradation. Contrarily, overexpression of NCOA4 promoted chondrocyte ferroptosis and the delivery of Ncoa4 adeno-associated virus 9 into knee joint of mice aggravated post-traumatic OA. Mechanistic study revealed that NCOA4 was upregulated in a JNK-JUN signaling-dependent manner in which JUN could directly bind to the promoter of Ncoa4 and initial the transcription of Ncoa4. NCOA4 could interact with ferritin and increase autophagic degradation of ferritin and iron levels, which caused chondrocyte ferroptosis and extracellular matrix degradation. In addition, inhibition of JNK-JUN-NCOA4 axis by SP600125, a specific inhibitor of JNK, attenuated development of post-traumatic OA. This work highlights the role of JNK-JUN-NCOA4 axis and ferritinophagy in chondrocyte ferroptosis and OA pathogenesis, suggesting this axis as a potential target for OA treatment.

ANLN Serves as an Oncogene in Bladder Urothelial Carcinoma via Activating JNK Signaling Pathway.

INTRODUCTION: To understand the significance of ANLN (anillin, actin-binding protein)-mediated c-Jun N-terminal kinase (JNK) signal pathway on the progression of bladder urothelial carcinoma (BLCA). METHODS: The Cancer Genome Atlas (TCGA) database was utilized to perform the clinical significance of ANLN in BLCA. Then, ANLN expression was determined in human normal primary bladder epithelial cells (BdEC) and BLCA cells. Later, ANLN knockdown was performed in BLCA cells, where the expression of MAPK8, MAPK9, and p-JNK/JNK was detected. BLCA cells were divided into the Mock, siNC, siANLN, SP600125 (a selective JNK inhibitor), and ANLN + SP600125 group, followed by measurements of real-time quantitative polymerase chain reaction, 3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Annexin V-FITC/PI, Wound-healing, Transwell, and immunofluorescence assays. RESULTS: ANLN was upregulated in the BLCA tissues, which showed a relation with the stage of patients. Besides, BLCA patients with high expression of ANLN had a worse prognosis than those with low expression of ANLN. Besides, the expression of ANLN in the BLCA tissues was positively correlated with MAPK8 and MAPK9. SP600125 suppressed the JNK signal pathway, reduced the proliferation, and increased BLCA cell apoptosis, with the reductions in the invasion and migration and the upregulation of phospho-histone H3 Ser-10 (pHH3), which was abolished by the overexpression of ANLN. CONCLUSION: ANLN, as an oncogene of BLCA, may associate with the activation of JNK signal pathway. Inhibiting ANLN could deactivate the JNK signal pathway, thereby suppressing the proliferation, invasion, and migration while promoting the apoptosis of BLCA cells.

On the effect of hippocampal c-Jun N-terminal kinase inhibition on object recognition memory.

c-Jun N-terminal kinase (JNK) phosphorylates the transcription factor c-Jun in response to stress stimuli and contributes to both hippocampal synaptic plasticity and memory processing in mammals. Object recognition memory (ORM) is essential for remembering facts and events. In rodents, ORM consolidation and reconsolidation require a functional hippocampus. However, the possible involvement of hippocampal JNK on ORM processing has not yet been studied. Here we show that when injected into dorsal CA1 5 min, but not 6 h, after training adult male rats in the novel object recognition learning task, the JNK inhibitor SP600125 impaired ORM for at least 7 days without affecting exploratory activity, short-term ORM retention, or the functional integrity of the hippocampus. SP600125 did not hinder ORM retention when given in CA1 after a memory reactivation session carried out 24 h post-training in the presence of the same two objects presented during the training session, but caused time-dependent amnesia when one of the objects presented at training was replaced by a different but behaviorally equivalent novel one. Taken together, our results indicate that hippocampal JNK activity is necessary for ORM consolidation and reconsolidation but not for ORM recall or short-term retention.

SP600125 Enhances Temperature-Controlled Repeated Thermal Stimulation-Induced Neurite Outgrowth in PC12-P1F1 Cells.

This study evaluated the mechanism of temperature-controlled repeated thermal stimulation (TRTS)-mediated neuronal differentiation. We assessed the effect of SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, on neuronal differentiation of rat PC12-P1F1 cells, which can differentiate into neuron-like cells by exposure to TRTS or neurotrophic factors, including bone morphogenetic protein (BMP) 4. We evaluated neuritogenesis by incubating the cells under conditions of TRTS and/or SP600125. Cotreatment with SP600125 significantly enhanced TRTS-mediated neuritogenesis, whereas that with other selective mitogen-activated protein kinase (MAPK) inhibitors did not-e.g., extracellular signal-regulated kinase (ERK)1/2 inhibitor U0126, and p38 MAPK inhibitor SB203580. We tried to clarify the mechanism of SP600125 action by testing the effect of U0126 and the BMP receptor inhibitor LDN193189 on the SP600125-mediated enhancement of intracellular signaling. SP600125-enhanced TRTS-induced neuritogenesis was significantly inhibited by U0126 or LDN193189. Gene expression analysis revealed that TRTS significantly increased ß3-Tubulin, MKK3, and Smad7 gene expressions. Additionally, Smad6 and Smad7 gene expressions were substantially attenuated through SP600125 co-treatment during TRTS. Therefore, SP600125 may partly enhance TRTS-induced neuritogenesis by attenuating the negative feedback loop of BMP signaling. Further investigation of the mechanisms underlying the effect of SP600125 during TRTS-mediated neuritogenesis may contribute to the future development of regenerative neuromedicine.

Blocking the cytohesin-2/ARF1 axis by SecinH3 ameliorates osteoclast-induced bone loss via attenuating JNK-mediated IRE1 endoribonuclease activity.

cytohesin-2 is a guanine nucleotide exchange factor to activate ARF1 and ARF6, which are involved in various biological processes, including signal transduction, cell differentiation, cell structure organization, and survival. Nevertheless, there is a lack of evidence revealing the role of cytohesin-2 in osteoclast differentiation and in the development of osteoporosis. In this study, we find cytohesin-2 and ARF1 positively regulate osteoclast differentiation and function. Blocking the cytohesin-2 /ARF1 axis with SecinH3 or by genetic silencing of cytohesin-2 inhibits osteoclast formation and function in vitro. In vivo treatment with SecinH3 ameliorates ovariectomy-induced osteoporosis. Mechanistically, RNA-sequencing combined with molecular biological methodologies reveal that the regulatory function of cythohesin-2/ARF1 axis in osteoclast differentiation is mainly dependent on activating the JNK pathway. Further, in addition to the common viewpoint that JNK is activated by IRE1 via its kinase activity, we found that JNK can act upstream and regulate the endoribonuclease activity of IRE1 to promote XBP1 splicing. Both SecinH3 and silencing of cytohesin-2 inhibit JNK activation and IRE1 endoribonuclease activity, leading to the suppression of osteoclast differentiation. Taken together, our findings add new insights into the regulation between JNK and IRE1, and reveal that inhibiting the cytohesin-2/ARF1/JNK/IRE1 axis might represent a potential new strategy for the treatment of post-menopause osteoporosis.

Carveol ameliorates mercury-induced oxidative stress, neuroinflammation, and neurodegeneration in a mouse brain.

BACKGROUND: Mercury compounds are the world's third most hazardous substance. Mercury (II) chloride, also known as mercuric chloride (HgCl2), has been shown to have neurotoxic properties in a variety of forms. In numerous investigations, oxidative stress has been established as a key contributor to HgCl2-induced neurotoxicity. Carveol has been researched as an antioxidant and Nrf2-activator in several studies. This study was conducted to investigate if the carveol could protect mice against HgCl2-induced neuronal damage. METHODS: Mice were exposed to a dose of 0.4 mg/kg of HgCl2 and 20 mg/kg of carveol for 21 days. Animals were then subjected to behavioral evaluation through various methods such as open field test (OFT), elevated plus maze test (EPM), morris-water maze test (MWM), and Y-maze test. RESULTS: Results indicated hippocampal-related behavior anomalies which were improved significantly after carveol treatment. Oxidative stress was accompanied by excessive neuroinflammation, which was demonstrated by elevated levels of inflammatory markers such as TNF-α, p-NFkB, and COX-2, and were measured by Western blot, ELISA, and immunohistochemistry. These elevated levels of inflammatory markers were significantly mitigated upon treatment with carveol. To further investigate the participation of the JNK pathway, we used SP-600125 to inhibit JNK, which enhanced the neuroprotective effects of carveol. Moreover, molecular docking and modeling studies were used to validate these effects. CONCLUSION: Our findings indicate that carveol can inhibit the p-JNK pathway, thereby inhibiting HgCl2-induced apoptosis and downregulating the expression of inflammatory mediators.

Inhibition of HDAC and Signal Transduction Pathways Induces Tight Junctions and Promotes Differentiation in p63-Positive Salivary Duct Adenocarcinoma.

BACKGROUND: The p53 family p63 is essential for the proliferation and differentiation of various epithelial basal cells. It is overexpressed in several cancers, including salivary gland neoplasia. Histone deacetylases (HDACs) are thought to play a crucial role in carcinogenesis, and HDAC inhibitors downregulate p63 expression in cancers. METHODS: In the present study, to investigate the roles and regulation of p63 in salivary duct adenocarcinoma (SDC), human SDC cell line A253 was transfected with siRNA-p63 or treated with the HDAC inhibitors trichostatin A (TSA) and quisinostat (JNJ-26481585). RESULTS: In a DNA array, the knockdown of p63 markedly induced mRNAs of the tight junction (TJ) proteins cingulin (CGN) and zonula occuludin-3 (ZO-3). The knockdown of p63 resulted in the recruitment of the TJ proteins, the angulin-1/lipolysis-stimulated lipoprotein receptor (LSR), occludin (OCLN), CGN, and ZO-3 at the membranes, preventing cell proliferation, and leading to increased cell metabolism. Treatment with HDAC inhibitors downregulated the expression of p63, induced TJ structures, recruited the TJ proteins, increased the epithelial barrier function, and prevented cell proliferation and migration. CONCLUSIONS: p63 is not only a diagnostic marker of salivary gland neoplasia, but it also promotes the malignancy. Inhibition of HDAC and signal transduction pathways is, therefore, useful in therapy for p63-positive SDC cells.

Anti-high-mobility group box-1 (HMGB1) mediates the apoptosis of alveolar epithelial cells (AEC) by receptor of advanced glycation end-products (RAGE)/c-Jun N-terminal kinase (JNK) pathway in the rats of crush injuries.

OBJECTIVES: The lung injury is often secondary to severe trauma. In the model of crush syndrome, there may be secondary lung injury. We hypothesize that high-mobility group box 1 (HMGB1), released from muscle tissue, mediates the apoptosis of alveolar epithelial cells (AEC) via HMGB1/Receptor of advanced glycation end-products (RAGE)/c-Jun N-terminal kinase (JNK) pathway. The study aimed to investigate how HMGB1 mediated the apoptosis of AEC in the rat model. METHODS: Seventy-five SD male rats were randomly divided into five groups: CS, CS + vehicle, CS + Ethyl pyruvate (EP), CS + FPS-ZM1 group, and CS + SP600125 groups. When the rats CS model were completed after 24 h, the rats were sacrificed. We collected the serum and the whole lung tissues. Inflammatory cytokines were measured in serum samples. Western blot and RT-qPCR were used to quantify the protein and mRNA. Lastly, apoptotic cells were detected by TUNEL. We used SPSS 25.0 for statistical analyses. RESULTS: Nine rats died during the experiments. Dead rats were excluded from further analysis. Compared to the CS group, levels of HMGB1 and inflammatory cytokines in serum were downregulated in CS + EP, CS + FPS-ZM1, and CS + SP600125 groups. Western blot and RT-qPCR analysis revealed a significant downregulation of HMGB1, RAGE, and phosphorylated-JNK in CS + EP, CS + FPS-ZM1, and CS + SP600125 groups, compared with the CS groups, excluding total-JNK mRNA. Apoptosis of AEC was used TUNEL to assess. We found the TUNEL-positive cells were downregulated in CS + EP, CS + FPS-ZM1, and CS + SP600125 groups. CONCLUSION: The remote lung injury begins early after crush injuries. The HMGB1/RAGE/JNK signaling axis is an attractive target to abrogate the apoptosis of AEC after crush injuries.

Inhibition of c-Jun N-terminal kinase signaling promotes osteoblastic differentiation of periodontal ligament stem cells and induces regeneration of periodontal tissues.

OBJECTIVES: Few clinical treatments to regenerate periodontal tissue lost due to severe endodontic and periodontal disease have yet been developed. Therefore, the development of new treatment methods for the regeneration of periodontal tissue is expected. The purpose of this study was to investigate the effects of a c-Jun N-terminal kinase (JNK) inhibitor, SP600125, on the osteoblastic differentiation of periodontal ligament stem cells (PDLSCs) in vitro, and the function of SP600125 on the regeneration of alveolar bone in vivo. DESIGN: Alizarin red S staining, quantitative RT-PCR, and western blotting analysis was performed to determine whether SP600125 affects osteoblastic differentiation of human PDLSCs (HPDLSCs) and bone-related intracellular signaling. The effect of SP600125 on the regeneration of alveolar bone was assessed by using a rat periodontal defect model. The healing of periodontal defects was evaluated using micro-CT scans and histological analysis. RESULTS: SP600125 promoted the osteoblastic differentiation such as Alizarin red S-positive mineralized nodule formation and the expression of osteoblast-related genes in HPDLSCs under osteogenic conditions. In addition, this inhibitor upregulated the BMP2 expression and the phosphorylation of Smad1/5/8 in HPDLSCs under the same conditions. The inhibition of Smad1/5/8 signaling by LDN193189 suppressed the SP600125-induced osteoblastic differentiation of HPDLSCs. Furthermore, the application of SP600125 promoted the regeneration of not only alveolar bone but also PDL tissue in periodontal defects. CONCLUSION: This study suggested that inhibition of JNK signaling promotes the osteoblastic differentiation of HPDLSCs through BMP2-Smad1/5/8 signaling, leading to the regeneration of periodontal tissues such as alveolar bone and PDL tissue.

Effects of SP600125 on Proliferation and Invasion of Human Cervical Cancer HeLa Cells / 肿瘤防治研究

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.

CpG Oligodeoxynucleotides Attenuate OVA-Induced Allergic Airway Inflammation via Suppressing JNK-Mediated Endoplasmic Reticulum Stress.

PURPOSE: CpG-ODN has been found to attenuate allergic airway inflammation in our previous study. Here, we aimed to further investigate whether CpG-ODN exerts such effect via regulating endoplasmic reticulum (ER) stress and revealed the underlying mechanism. METHODS: Five-week-old C57BL/6 mice were randomly grouped and treated with or without CpG-ODN or/and SP600125. Meantime, RAW264.7 cells were used to investigate the effect of CpG-ODN on OVA-induced ER stress in vitro. The cellularity of bronchoalveolar lavage fluid (BALF) was classified and counted after Wright-Giemsa staining. HE and PAS staining methods were applied to analyze airway inflammation. The protein levels of IL-4, IL-5, IL-13, p-JNK, JNK, CHOP, XBP1, ATF6α and GRP78 in lung tissues were detected by Western blotting. Correspondingly, the ER stress markers were detected by Western blotting and immunofluorescence in RAW264.7 cells. RESULTS: In OVA-induced allergic airway inflammation, CpG-ODN significantly suppressed inflammatory cells infiltration, goblet cell hyperplasia and the protein expression of Th2 cytokines. Moreover, OVA exposure strongly increased the activation of ER stress with higher protein expressions of CHOP, XBP1, ATF6α and GRP78. However, these OVA-induced increase of ER stress markers were markedly suppressed by CpG-ODN treatment. In addition, exposure to OVA significantly increased the phosphorylation of JNK, which was significantly reduced by CpG-ODN treatment. Remarkably, single treatment of SP600125, an antagonist of JNK, functioned similarly as CpG-ODN in mitigating allergic airway inflammation and suppressing OVA-induced activation of ER stress; however, no significant synergistic effect was evidenced by combined treatment of SP600125 and CpG-ODN. Furthermore, in OVA-stimulated RAW264.7 cells, we also found that OVA stimulation increased the expressions of ER stress markers, and CpG-ODN significantly reduced their expression levels via suppressing the phosphorylation of JNK. CONCLUSION: These results indicated that CpG-ODN mitigates allergic airway inflammation via suppressing the activation of JNK-medicated ER stress.

Characteristics of SP600125 Induced Tetraploid Cells in Comparison With Diploid and Tetraploid Cells of Fish.

In our previous research, SP600125 (Anthrapyrazolone) was used to induce autotetraploid of crucian carp cells (SP4N cells), and tetraploid fry was generated from the SP4N cells by somatic cell nuclear transfer technique. However, it is still unclear about biological characteristics of the SP4N cells. In this article, the cytological characteristic and gene expression profiles of the SP4N cells are investigated in comparison with the crucian carp cells (2N cells) and the tetraploid crucian carp cells (CC4N cells). The SP4N cells have tetraploid characteristics in terms of morphology and DNA ploidy levels, and their chromosome behavior is stable during the cell proliferation. The migration ability and the mtDNA copy number of SP4N cells are both lower than those in the CC4N cells and the 2N cells, but there exist giant mitochondria in the SP4N cells. The similar expression trends in the cell cycle regulation genes of the SP4N cells and 2N cells, while the corresponding expression profiles are clearly different between the SP4N cells and the CC4N cells. Moreover, the significant difference genes are associated with energy metabolism pathways among the SP4N cells, 2N cells and CC4N cells. These results can provide deeper understanding of SP600125 induction, as well as finding applications in polyploidization breeding of fish species.

SP600125 Restored TNF-α-Induced Impaired Chondrogenesis in Bone Mesenchymal Stem Cells and Its Antiosteoarthritis Effect in Mice.

Inflammation, the main factor in the progression of osteoarthritis (OA), impairs the chondrogenesis of bone mesenchymal stem cells (BMSCs), which is an appealing process to target to regenerate impaired articular cartilage. This article aimed to investigate whether SP600125, a competitive ATP-specific inhibitor of the JNK pathway, could promote the chondrogenesis of BMSCs by enhancing their anti-inflammatory capacity. Chondrogenic differentiation was assessed by Alcian blue staining, immunofluorescence staining, and Western blot. The inflammation level was associated with the expression of matrix metalloproteinases (Mmp), evaluated by Western blot. Intra-articular injection of BMSCs pretreated with or without SP600125 was carried out on C57BL/6 mice after inducing OA by surgical destabilization of the medial meniscus. Safranin O-fast green (SO) and hematoxylin-eosin staining were employed to evaluate the cartilage destruction and immunohistochemical analysis was adopted to detect the expression of Col2 and Mmp-13 proteins in the mouse knee joint. We showed that SP600125 could inhibit inflammation induced by tumor necrosis factor-α (TNF-α) and promote the chondrogenesis of BMSCs. In the presence of TNF-α, the expression of aggrecan (Agc) and collagen type II alpha 1 (Col2) was significantly decreased compared with that in the control group and increased with the addition of SP600125. Moreover, the expression of Mmp-1, Mmp-3, and Mmp-13 was increased in BMSCs treated only with TNF-α and downregulated in SP600125-treated BMSCs. In vivo study showed that SP600125 could enhance protective effects of BMSCs on OA mice. Our results indicated that SP600125 rescued the chondrogenesis of BMSCs by inhibiting inflammation induced by TNF-α, which provides a theoretical basis for solving the problem of cartilage repair under inflammatory conditions.

Effect of Dickkopf-1 (Dkk-1) and SP600125, a JNK Inhibitor, on Wnt Signaling in Canine Prostate Cancer Growth and Bone Metastases.

Human Dickkopf-1 (Dkk-1) upregulates a noncanonical Wnt/JNK pathway, resulting in osteoclast stimulation, cell proliferation, and epithelial-to-mesenchymal transition (EMT) of cancer cells. Ace-1-Dkk-1, a canine prostate cancer (PCa) cell line overexpressing Dkk-1, was used to investigate Wnt signaling pathways in PCa tumor growth. SP600125, a JNK inhibitor, was used to examine whether it would decrease tumor growth and bone tumor phenotype in canine PCa cells in vitro and in vivo. Ace-1-VectorYFP-Luc and Ace-1-Dkk-1YFP-Luc cells were transplanted subcutaneously, while Ace-1-Dkk-1YFP-Luc was transplanted intratibially into nude mice. The effects of Dkk-1 and SP600125 on cell proliferation, in vivo tumor growth, and bone tumor phenotype were investigated. The mRNA expression levels of Wnt/JNK-related genes were measured using RT-qPCR. Dkk-1 significantly increased the mRNA expression of Wnt/JNK-signaling-related genes. SP600125 significantly upregulated the mRNA expression of osteoblast differentiation genes and downregulated osteoclastic-bone-lysis-related genes in vitro. SP600125 significantly decreased tumor volume and induced spindle-shaped tumor cells in vivo. Mice bearing intratibial tumors had increased radiographic density of the intramedullary new bone, large foci of osteolysis, and increased cortical lysis with abundant periosteal new bone formation. Finally, SP600125 has the potential to serve as an alternative adjuvant therapy in some early-stage PCa patients, especially those with high Dkk-1 expression.

Antidepressant effect of electroacupuncture on modulating the expression of c-Fos/AP-1 through the JNK signaling pathway.

The effectiveness and safety of electroacupuncture (EA) for depression have been identified by abundant clinical trials and experimental findings. The c-Jun-NH(2)-terminal kinase (JNK) signaling pathway is considered to be involved in the antidepressant mechanism of EA. However, the antidepressant effect of EA via modulating the expression of c-Fos/activator protein-1 (AP-1) under the condition of JNK inhibition remains unexplored. In this study, we investigated the antidepressant effect and possible mechanism of EA in regulating the expression of c-Fos/AP-1 under the condition of JNK inhibition by SP600125 in rats exposed to chronic unpredictable mild stress (CUMS). The depression-like behaviors were evaluated by the body weight, sucrose preference test (SPT), and open field test (OFT). The expression levels of c-Jun in the hypothalamus, c-Fos in the pituitary gland, and c-Fos and AP-1 in the serum of CUMS induced rat model of depression were detected by ELISA. The results indicated that treatment with EA and fluoxetine can reverse the CUMS-induced depression-like behaviors in rats and can up-regulate the expression levels of c-Jun in the hypothalamus, c-Fos in the pituitary gland, and c-Fos and AP-1 in the serum. Of note, the data demonstrated that SP600125, the inhibitor of JNK signaling pathway, can exert synergistic effect with EA in regulating CUMS-induced abnormal activation of the JNK signaling pathway. The antidepressant effect of EA might be mediated by modulating the expression of c-Fos/AP-1.