A Dual Target-Directed Single Domain-Based Fusion Protein Against Interleukin-6 Receptor Decelerate Experimental Arthritis Progression Via Modulating JNK Expression.

The currently used anti-cytokine therapeutic antibodies cannot selectively neutralize pathogenic cytokine signalling that cause collateral damage to protective signalling cascades. The single domain chain firstly discovered in Camelidae displays fully functional ability in antigen-binding against variable targets, which has been seemed as attractive candidates for the next-generation biologic drug study. In this study, we established a simple prokaryotic expression system for a dual target-directed single domain-based fusion protein against the interleukin-6 receptor and human serum, albumin, the recombinant anti-IL-6R fusion protein (VHH-0031). VHH-0031 exhibited potent anti-inflammatory effects produced by LPS on cell RAW264.7, where the major cytokines and NO production were downregulated after 24 h incubation with VHH-0031 in a dose-dependent manner. In vivo, VHH-0031 presented significant effects on the degree reduction of joint swelling in the adjuvant-induced arthritis (AIA) rat, having a healthier appearance compared with the dexamethasone. The expression level of JNK protein in the VHH-0031 group was significantly decreased, demonstrating that VHH-0031 provides a low-cost and desirable effect in the treatment of more widely patients.

MicroRNA-27a-3p Downregulation Inhibits Inflammatory Response and Hippocampal Neuronal Cell Apoptosis by Upregulating Mitogen-Activated Protein Kinase 4 (MAP2K4) Expression in Epilepsy: In Vivo and In Vitro Studies.

BACKGROUND This study aimed to discover the effect and mechanism of microRNA-27a-3p (miR-27a-3p) in epilepsy. MATERIAL AND METHODS To perform our investigation, in vivo and in vitro models of epilepsy were induced using kainic acid (KA). Expression of miR-27a-3p in the hippocampus of epileptic rats or normal rats or neuronal cells was detected using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Racine score was used to assess seizures in epileptic rats. Cell viability and cell apoptosis were analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was performed to detect inflammatory factors expression. RESULTS Significantly higher expression of miR-27a-3p in the hippocampus of epileptic rats and in KA-induced neurons was observed. We found that miR-27a-3p inhibitor alleviated seizures in epileptic rats. miR-27a-3p inhibitor also inhibited apoptosis of hippocampal neurons in epileptic rats, promoted Bcl2 expression, and decreased Bax and Caspase3 expression. The results showed that miR-27a-3p inhibitor effectively reduced the expression levels of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-alpha (TNF-alpha) in hippocampal tissues of epileptic rats. Dual luciferase reporter assay showed that mitogen-activated protein kinase 4 (MAP2K4) was a direct target of miR-27a-3p. miR-27a-3p inhibitor significantly promoted the cell viability of KA-induced neurons, inhibited cell apoptosis, promoted the expression of Bcl-2, and decreased Bax and Caspase3 expression, and all these changes were abolished by MAP2K4-siRNA co-transfection. CONCLUSIONS Our preliminary findings indicated that miR-27a-3p inhibitor protected against epilepsy-induced inflammatory response and hippocampal neuronal apoptosis by targeting MAP2K4.

Similar Aquaporin9 and MAPK Expression Profiles in the Liver of Types 1 and 2 Diabetes Mellitus.

Aquaporin-9 (AQP9) is an aquaglyceroporin that biophysically conducts water, glycerol, and other small solutes. AQP9 is expressed in hepatocytes on the sinusoidal surfaces of hepatocyte plates in the liver, where it is considered responsible for the glycerol uptake in gluconeogenesis. However, limited information is available on the expression and regulating mechanism of AQP9 in different hyperglycemia models. Thus, this study examined the expression patterns of AQP9 and mitogen-activated protein kinase (MAPK) in Types 1 and 2 diabetes mellitus (DM) to clarify the roles and regulating mechanism of AQP9 in gluconeogenesis. Compared with the control group, the AQP9 expression significantly increased in both Types 1 and 2 DM, and the increased expression was associated with the activation of phosphorylated JNK (p-JNK) and the inhibition of phosphorylated p38 (p-p38). By contrast, phosphorylated ERK remained stable in the liver with Type 1 or 2 DM. These effects could be reversed by insulin treatment. That is, insulin downregulated AQP9 by inhibiting p-JNK and activating p-p38. The upregulation of AQP9 could be involved in gluconeogenesis and co-regulated by the JNK and p38 MAPK pathway in both Types 1 and 2 DM.

Lactic Acid Downregulates Viral MicroRNA To Promote Epstein-Barr Virus-Immortalized B Lymphoblastic Cell Adhesion and Growth.

High plasma lactate is associated with poor prognosis of many malignancies, but its role in virally mediated cancer progression and underlying molecular mechanisms are unclear. Epstein-Barr virus (EBV), the first human oncogenic virus, causes several cancers, including B-cell lymphoma. Here, we report that lactate dehydrogenase A (LDH-A) expression and lactate production are elevated in EBV-immortalized B lymphoblastic cells, and lactic acid (LA; acidic lactate) at low concentration triggers EBV-infected B-cell adhesion, morphological changes, and proliferation in vitro and in vivo Moreover, LA-induced responses of EBV-infected B cells uniquely occurs in viral latency type III, and it is dramatically associated with the inhibition of global viral microRNAs, particularly the miR-BHRF1 cluster, and the high expression of SMAD3, JUN, and COL1A genes. The introduction of miR-BHRF1-1 blocks the LA-induced effects of EBV-infected B cells. Thus, this may be a novel mechanism to explain EBV-immortalized B lymphoblastic cell malignancy in an LA microenvironment.IMPORTANCE The tumor microenvironment is complicated, and lactate, which is created by cell metabolism, contributes to an acidic microenvironment that facilitates cancer progression. However, how LA operates in virus-associated cancers is unclear. Thus, we studied how EBV (the first tumor virus identified in humans; it is associated with many cancers) upregulates the expression of LDH-A and lactate production in B lymphoma cells. Elevated LA induces adhesion and the growth of EBV-infected B cells by inhibiting viral microRNA transcription. Thus, we offer a novel understanding of how EBV utilizes an acidic microenvironment to promote cancer development.

Tumor necrosis factor alpha-stimulated gene-6 (TSG-6) inhibits the inflammatory response by inhibiting the activation of P38 and JNK signaling pathway and decreases the restenosis of vein grafts in rats.

This study aims to explore the effects of tumor necrosis factor alpha-stimulated gene-6 (TSG-6) on vascular inflammatory response and vascular injury in grafted vein wall of rats and its possible mechanism. Vascular grafting model was established by modified cuff. The effect of TSG-6 on the inflammatory response and vascular injury of vein graft was investigated. The activation of mast cells and macrophages after LPS stimulation was observed by lentivirus-mediated upregulation or downregulation of TSG-6 expression. The results showed that rhTSG-6 treatment could significantly inhibit the proliferation of venous bridge, decrease macrophage infiltration and smooth muscle cell proliferation. The expression levels of TNF-α and IL-1 in treated group were significantly lower than that of untreated group (P < 0.05), while the expression of IL-10 in treated group were significantly higher than that of untreated group (P < 0.05). The expression levels of P38, p-P38, JNK and p-JNK in venous bridge of rats were significantly lower than those of untreated rats (P < 0.05), while there was no significant difference in the expression level of ERK and p-ERK (P > 0.05). TSG-6 could inhibit the proliferation of mast cells and macrophages and the release of inflammatory cytokines by down regulating the expression levels of P38, p-P38, JNK and p-JNK. TSG-6 can inhibit the inflammatory response of transplanted vein grafts in rats and reduce vascular injury by downregulation of P38 and JNK signaling pathway.

IDH1 Mutation Promotes Tumorigenesis by Inhibiting JNK Activation and Apoptosis Induced by Serum Starvation.

Two hallmarks of cancer cells are their resistance to apoptosis and ability to thrive despite reduced levels of vital serum components. c-jun N-terminal kinase (JNK) activation is crucial for apoptosis triggered by serum starvation (SS), and isocitrate dehydrogenase 1 (IDH1) mutations are tumorigenic, in part, because they produce the abnormal metabolite 2-hydroxyglutarate (2-HG). However, it is unknown whether 2-HG-induced tumorigenesis is partially due to JNK inhibition and thus defective SS-induced apoptosis. We show here, using IDH1-R132Q knockin mutant mouse cells, that 2-HG inhibits JNK activation induced only by SS and not by UV or doxorubicin, and thus can block apoptosis. Upon SS, Cdc42 normally disrupts mixed lineage kinase 3's (MLK3's) auto-inhibition, triggering the MLK3-MKK4/7-JNK-Bim apoptotic cascade. 2-HG binds to Cdc42 and abolishes its association with MLK3, inactivating MLK3 and apoptosis. Allograft tumor assays in mice demonstrate that this mechanism contributes to tumorigenesis driven by mutant IDH1, a result confirmed by detection of JNK inactivation in human gliomas harboring IDH1-R132H mutations.

Psoralen Inhibited Apoptosis of Osteoporotic Osteoblasts by Modulating IRE1-ASK1-JNK Pathway.

Osteoporosis is a common disease causing fracture in older populations. Abnormal apoptosis of osteoblasts contributes to the genesis of osteoporosis. Inhibiting apoptosis of osteoblasts provides a promising strategy to prevent osteoporosis. The proliferation of osteoblasts isolated from osteoporotic patients or healthy subjects was determined by MTT assay. Apoptosis was determined by Annexin V/PI assay. Protein expression was measured by western blot. The proliferation of osteoblasts isolated from osteoporotic patients was inhibited and the apoptosis level of these cells was higher than the osteoblasts from healthy subjects. Incubation with psoralen or estradiol significantly enhanced the proliferation and decreased the apoptosis level of osteoporotic osteoblasts. Western blot demonstrated that psoralen or estradiol treatment downregulated the expression of IRE1, p-ASK, p-JNK, and Bax. Meanwhile, expression of Bcl-2 was upregulated. Pretreatment by IRE1 agonist tunicamycin or JNK agonist anisomycin attenuated the effect of psoralen on osteoporotic osteoblasts. Psoralen inhibited apoptosis of osteoporotic osteoblasts by regulating IRE1-ASK1-JNK pathway.

MicroRNA-802 plays a tumour suppressive role in tongue squamous cell carcinoma through directly targeting MAP2K4.

OBJECTIVES: Tongue squamous cell carcinoma (TSCC) is the most common oral tumours. MicroRNAs play crucial roles in many cell processes including cell viability, development, apoptosis, migration and invasion. The role of miR-802 in the TSCC is still unknown. MATERIALS AND METHODS: The miR-802 expression in TSCC tissues and cell lines was determined by quantitative real-time polymerase chain reaction. CCK-8 assay was performed to measure the cell viability, while the cell invasion assay was used to determine the cell invasion. Dual-luciferase reporter and western blot were used to confirm the potential target gene of miR-802. RESULTS: In our study, we demonstrated that miR-802 expression was downregulated in TSCC tissues and cell lines. Elevated expression of miR-802 suppressed the TSCC cell viability and invasion. Moreover, enforced expression of miR-802 increased the expression of E-cadherin, while suppressed the expression of N-cadherin, Snail and Vimentin in the TSCC cell. In addition, we identified the mitogen-activated protein kinase 4 (MAP2K4) as a direct target gene of miR-802 in the TSCC cell. We also demonstrated that the expression of MAP2K4 was higher in the TSCC tissues than that in the adjacent normal tissues. Furthermore, the expression level of MAP2K4 was inversely associated with the expression of miR-802 in TSCC tissues. We also demonstrated that the MAP2K4 expression was upregulated in TSCC cell lines. Elevated expression of miR-802 inhibited TSCC cell viability and invasion through inhibiting MAP2K4 expression. CONCLUSIONS: Our data revealed that miR-802 played as a tumour suppressor gene and might act as a therapeutic target in TSCC patients.

Mitochondria and Caspases Tune Nmnat-Mediated Stabilization to Promote Axon Regeneration.

Axon injury can lead to several cell survival responses including increased stability and axon regeneration. Using an accessible Drosophila model system, we investigated the regulation of injury responses and their relationship. Axon injury stabilizes the rest of the cell, including the entire dendrite arbor. After axon injury we found mitochondrial fission in dendrites was upregulated, and that reducing fission increased stabilization or neuroprotection (NP). Thus axon injury seems to both turn on NP, but also dampen it by activating mitochondrial fission. We also identified caspases as negative regulators of axon injury-mediated NP, so mitochondrial fission could control NP through caspase activation. In addition to negative regulators of NP, we found that nicotinamide mononucleotide adenylyltransferase (Nmnat) is absolutely required for this type of NP. Increased microtubule dynamics, which has previously been associated with NP, required Nmnat. Indeed Nmnat overexpression was sufficient to induce NP and increase microtubule dynamics in the absence of axon injury. DLK, JNK and fos were also required for NP. Because NP occurs before axon regeneration, and NP seems to be actively downregulated, we tested whether excessive NP might inhibit regeneration. Indeed both Nmnat overexpression and caspase reduction reduced regeneration. In addition, overexpression of fos or JNK extended the timecourse of NP and dampened regeneration in a Nmnat-dependent manner. These data suggest that NP and regeneration are conflicting responses to axon injury, and that therapeutic strategies that boost NP may reduce regeneration.

Tropisetron Protects Against Acetaminophen-Induced Liver Injury via Suppressing Hepatic Oxidative Stress and Modulating the Activation of JNK/ERK MAPK Pathways.

Objectives. To investigate the protective effects of tropisetron on acetaminophen- (APAP-) induced liver injury in a mice model. Methods. C57BL/6 male mice were given tropisetron (0.3 to 10 mg/kg) 30 minutes before a hepatotoxic dose of acetaminophen (300 mg/kg) intraperitoneally. Twenty hours after APAP intoxication, sera alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, hepatic myeloperoxidase (MPO), malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) activities, and liver histopathological changes were examined. The MAP kinases were also detected by western blotting. Results. Our results showed that tropisetron pretreatment significantly attenuated the acute elevations of the liver enzyme ALT level, hepatic MPO activity, and hepatocytes necrosis in a dose-dependent manner (0.3-10 mg/kg) in APAP-induced hepatotoxicity mice. Tropisetron (1 and 3 mg/kg) suppressed APAP-induced hepatic lipid peroxidation expression and alleviated GSH and SOD depletion. Administration of tropisetron also attenuated the phosphorylation of c-Jun-NH2-terminal protein kinase (JNK) and extracellular signal-regulated kinase (ERK) caused by APAP. Conclusion. Our data demonstrated that tropisetron's hepatoprotective effect was in part correlated with the antioxidant, which were mediated via JNK and ERK pathways on acetaminophen-induced liver injury in mice.

ROCK activity affects IL-1-induced signaling possibly through MKK4 and p38 MAPK in Caco-2 cells.

Elevated levels of interleukin-1 (IL-1) accompany inflammatory bowel disease. IL-1-stimulated intestinal epithelial cells can secrete potent chemokines like CXCL8 to exacerbate inflammation. Previously, we found that inhibiting the Rho-associated kinase (ROCK) could inhibit IL-1- or TNF-α-induced CXCL8 secretion by the Caco-2 colonic epithelial cell line. This ROCK inhibition did not affect IκBα phosphorylation and degradation, but suppressed the phosphorylation of c-Jun N-terminal kinase (JNK). Therefore, ROCK must play an important role in epithelial cell CXCL8 responses through an effect on the JNK signaling pathway. Here, we extend these studies by showing that inhibiting ROCK suppressed the IL-1-induced phosphorylation of MKK4, a known activator of JNK, but not MKK7. Yet, ROCK inhibition had no significant effect on the IL-1-induced phosphorylation of extracellular-signal-regulated kinase (ERK) 1/2. Inhibiting ROCK also suppressed the phosphorylation of p38 MAPK after IL-1 stimulation, but this inhibition had no significant effect on the stability of CXCL8 messenger RNA (mRNA) after IL-1 stimulation. These results suggest that ROCK may be important in IL-1-induced signaling through MKK4 to JNK and the activation of p38 MAPK. Finally, inhibiting ROCK in IL-1 and TNF-α co-stimulated Caco-2 cells also resulted in a significant suppression of CXCL8 secretion and mRNA levels suggesting that inhibiting ROCK may be a mechanism to inhibit the overall response of epithelial cells to both cytokines. These studies indicate a novel signaling event, which could provide a target for suppressing intestinal epithelial cells (IEC) chemokine responses involved in mucosal inflammation.

Mimosine suppresses the PGF2α-induced synthesis of osteoprotegerin but not interleukin-6 in osteoblasts.

Mimosine, a plant amino acid, is known to act as a normoxic inducer of hypoxia-inducible factor (HIF). Previous research has suggested that HIF plays important roles in angiogenesis-osteogenesis coupling and bone metabolism. We previously reported that prostaglandin F2α (PGF2α) induced osteoprotegerin synthesis through p38 mitogen-activated protein (MAP) kinase, p44/p42 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. We have also demonstrated that PGF2α induced the synthesis of interleukin-6 (IL-6) via p38 MAP kinase and p44/p42 MAP kinase but not SAPK/JNK in these cells. In the present study, we investigated the effects of mimosine on the PGF2α-induced synthesis of osteoprotegerin or IL-6 in MC3T3-E1 cells. We found that deferoxamine, another inducer of HIF, as well as mimosine, upregulated the protein levels of HIF-1α. Both mimosine and deferoxamine significantly suppressed the PGF2α-induced release of osteoprotegerin, and the mRNA expression level, without markedly affecting PGF2α-induced IL-6 release. Both mimosine and deferoxamine, by themselves, induced the release of vascular endothelial growth factor. The phosphorylation of p38 MAP kinase, p44/p42 MAP kinase or SAPK/JNK induced by PGF2α was not markedly affected by either mimosine or deferoxamine. Thus, the results of the present study strongly suggest that mimosine, a normoxic inducer of HIF, inhibits the PGF2α­induced osteoprotegerin synthesis without affecting the IL-6 synthesis in osteoblasts.

Curcumin enhances the antitumor effect of ABT-737 via activation of the ROS-ASK1-JNK pathway in hepatocellular carcinoma cells.

At present, the therapeutic treatment strategies for patients with hepatocellular carcinoma (HCC) remain unsatisfactory, and novel methods are urgently required to treat this disease. Members of the B cell lymphoma (Bcl)-2 family are anti­apoptotic proteins, which are commonly expressed at high levels in certain HCC tissues and positively correlate with the treatment resistance of patients with HCC. ABT-737, an inhibitor of Bcl-2 anti-apoptotic proteins, has been demonstrated to exhibit potent antitumor effects in several types of tumor, including HCC. However, treatment with ABT-737 alone also activates certain pro-survival signaling pathways, which attenuate the antitumor validity of ABT-737. Curcumin, which is obtained from Curcuma longa, is also an antitumor potentiator in multiple types of cancer. In the present study, the synergistic effect of curcumin and ABT-737 on HCC cells was investigated for the first time, to the best of our knowledge. It was found that curcumin markedly enhanced the antitumor effects of ABT-737 on HepG2 cells, which was partially dependent on the induction of apoptosis, according to western blot analysis and flow cytometric apoptosis analysis. In addition, the sustained activation of the ROS-ASK1-c-Jun N-terminal kinase pathway may be an important mediator of the synergistic effect of curcumin and ABT-737. Collectively, these results indicated that the combination of curcumin and ABT-737 can efficaciously induce the death of HCC cells, and may offer a potential treatment strategy for patients with HCC.

N­trans­ρ­caffeoyl tyramine isolated from Tribulus terrestris exerts anti­inflammatory effects in lipopolysaccharide­stimulated RAW 264.7 cells.

Inflammation is induced by the expression of cyclooxygenase­2 (COX­2), which is an important mediator of chronic inflammatory diseases, such as rheumatoid arthritis, asthma and inflammatory bowel disease. Tribulus terrestris (T. terrestris) is known to have a beneficial effect on inflammatory diseases. In this study, we investigated the effects of N­trans­ρ­caffeoyl tyramine (CT) isolated from T. terrestris on the production of nitric oxide (NO), and the expression of pro­inflammatory cytokines and COX­2 in lipopolysaccharide (LPS)­stimulated RAW 264.7 cells. We also aimed to elucidate the molecular mechanisms involved. We found that the ethanolic extract of T. terrestris (EETT) and CT inhibited the production of NO, tumor necrosis factor­α (TNF­α), interleukin (IL)­6 and IL­10 in the LPS­stimulated RAW 264.7 cells in a dose­dependent manner. They were determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). In addition, CT markedly suppressed the expression of COX­2 and the production of prostaglandin E2 (PGE2) in response to LPS stimulation. Furthermore, CT markedly decreased p­c­Jun N­terminal kinase (p­JNK) protein expression in LPS­stimulated RAW 264.7 cells. COX-2 and p-JNK were measured by western blot analysis. Taken together, these findings indicate that CT isolated from T. terrestris is a novel and potent modulator of inflammatory responses. Thus, it may prove benefiical to further evaluate CT as a possible treatment for chronic inflammatory diseases.

Effect of N-n-butyl haloperidol iodide on ROS/JNK/Egr-1 signaling in H9c2 cells after hypoxia/reoxygenation.

Reactive oxygen species (ROS)-induced oxidative stress in cells is an important pathophysiological process during myocardial ischemia/reperfusion (I/R) injury, and the transcription factor Egr-1 is a master switch for various damage pathways during reperfusion injury. An in vitro model of myocardial I/R injury and H9c2 cardiomyoblast cells hypoxia/reoxygenation (H/R) was used to assess whether there is abnormal intracellular ROS/JNK/Egr-1 signaling. We also assessed whether N-n-butyl haloperidol (F2), which exerts protective effects during myocardial I/R injury, can modulate this pathway. H/R induced ROS generation, JNK activation, and increased the expression of Egr-1 protein in H9c2 cells. The ROS scavengers edaravone (EDA) and N-acetyl-L-cysteine (NAC) reduced ROS level, downregulated JNK activation, and Egr-1 expression in H9c2 cells after H/R. The JNK inhibitor SP600125 inhibited Egr-1 overexpression in H9c2 cells caused by H/R. F2 could downregulate H/R-induced ROS level, JNK activation, and Egr-1 expression in H9c2 cells in a dose-dependent manner. The ROS donor hypoxanthine-xanthine oxidase (XO/HX) and the JNK activator ANISO antagonized the effects of F2. Therefore, H/R activates ROS/Egr-1 signaling pathway in H9c2 cells, and JNK activation plays an important role in this pathway. F2 regulates H/R-induced ROS/JNK/Egr-1 signaling, which might be an important mechanism by which it antagonizes myocardial I/R injury.

Gartanin induces autophagy through JNK activation which extenuates caspase-dependent apoptosis.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Development of novel agents to eradicate liver cancer cells is required for treatment of HCC. Gartanin, a xanthone-type compound isolated from mangosteen, is known to possess potent antioxidant, anti-inflammatory, antifungal and antineoplastic properties. In the present study, we investigated the cytotoxic effect of gartanin on HCC and explored the cell death mechanism. We showed that gartanin induced both the extrinsic and intrinsic apoptotic pathways, which were interconnected by caspase-8, -9 and -3 activation. We also provided convincing evidence that gartanin induced autophagy in various cancer cells, as demonstrated by acridine orange staining of intracellular acidic vesicles, the degradation of p62, the conversion of LC3-I to LC3-II and GFP-LC3 punctate fluorescence. Additionally, gartanin induced the formation of typical autophagosomes and autolysosomes and enhanced the degradation rate of intracellular granule(s), including mitochondria. Notably, gartanin-mediated apoptotic cell death was further potentiated by pretreatment with autophagy inhibitors (3-methyladenine and bafilomycin A1) or small interfering RNAs against the autophagic genes (Atg5). These findings suggested that gartanin-mediated autophagic response protected against eventual cell death induced by gartanin. Moreover, gartanin treatment led to phosphorylation/activation of JNK and JNK-dependent phosphorylation of Bcl-2. Importantly, JNK inhibitor (SP600125) inhibited autophagy yet promoted gartanin-induced apoptosis, indicating a key requirement of the JNK-Bcl-2 pathway in the activation of autophagy by gartanin. Taken together, our data suggested that the JNK-Bcl-2 pathway was the critical regulator of gartanin-induced protective autophagy and a potential drug target for chemotherapeutic combination.

AMD3100 reduces CXCR4-mediated survival and metastasis of osteosarcoma by inhibiting JNK and Akt, but not p38 or Erk1/2, pathways in in vitro and mouse experiments.

Osteosarcoma (OS) has an unfavorable prognosis and tends to metastasize to lung tissue. Although the CXCL12-CXCR4 axis appears to affect progression and metastasis in numerous tumors, its mechanism and downstream pathways in OS remain unclear. We used western blotting and flow cytometry to detect CXCR4 and CXCR7 expression in two OS cell lines (LM8 and Dunn). An MTT assay was used to evaluate the effects of CXCL12 and AMD3100, a specific CXCR4 antagonist, on cell viability. Flow cytometry was utilized to analyze changes in apoptosis induced by serum deprivation following treatment with CXCL12 and AMD3100. A Transwell assay was used to assess cell migration in response to CXCL12 and AMD3100. Western blotting was performed to identify the phosphorylation of signaling molecules (JNK, c-Jun, Akt, p38 and Erk1/2) and expression of caspase-3 and -8, and PARP. Mouse models were employed to evaluate AMD3100 inhibition of primary OS growth and lung metastasis in vivo. CXCR4 expression was detected in LM8 but not Dunn cells, and neither cell line expressed CXCR7. The addition of CXCL12 induced the survival and migration of serum-starved CXCR4+ LM8 cells activating JNK and Akt pathways, which were abrogated by adding AMD3100. However, similar results were not observed in CXCR4- Dunn cells. CXCL12 protected LM8, but not Dunn cells, from apoptosis induced by serum deprivation by suppressing PARP cleavage, which was partly reversed by AMD3100. In a mouse model, AMD3100 reduced primary tumor growth and lung metastasis compared with the controls. Thus, the CXCL12-CXCR4 axis regulated OS survival and metastasis through the JNK and Akt pathways, and blocking them with AMD3100 was found to be a potential OS treatment.

Silencing of the tumor suppressor gene WNK2 is associated with upregulation of MMP2 and JNK in gliomas.

Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade extracellular matrix (ECM), thus assisting invasion. Upregulation of MMPs, frequently reported in gliomas, is associated with aggressive behavior. WNK2 is a tumor suppressor gene expressed in normal brain, and silenced by promoter methylation in gliomas. Patients without WNK2 exhibited poor prognosis, and its downregulation was associated with increased glioma cell invasion. Here we showed that MMP2 expression and activity are increased in glioma cell lines that do not express WNK2. Also, WNK2 inhibited JNK, a process associated with decreasing levels of MMP2. Thus, WNK2 promoter methylation and silencing in gliomas is associated with increased JNK activation and MMP2 expression and activity, thus explaining in part tumor cell invasion potential.

Studies on mitogen-activated protein kinase signaling pathway in the alveolar macrophages of chronic bronchitis rats.

Lipopolysaccharide (LPS), a potent stimulator of inflammatory responses in alveolar macrophages (AMs), activates several intracellular signaling pathways, including mitogen-activated protein kinases (MAPK). In the present study, we investigated the MAPK pathway in AMs of chronic bronchitis (CB) rats. CB was induced by endotracheal instillation of LPS followed by Bacillus Calmette Guerin injection through the caudal vein 1 week later. Specific inhibitors were used and protein phosphorylations were detected by Western blot. We found that Genistein (PTK inhibitor) could inhibit protein kinase C (PKC), phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt or PKB) MAPK signaling pathway with different degrees, LY294002 (PI3K inhibitor) could not only inhibit phospho-PI3K/Akt expression, but also inhibit p38 and c-Jun NH2-terminal kinases (JNK) phosphorylation. Calphostin C (PKC inhibitor) could inhibit phospho-PKC expression and exerted significant effects on extracellular signal-regulated kinases (ERK) phosphorylation, however, it had no impact on p38 and JNK phosphorylation. These results demonstrated that the LPS mediated signaling pathway of MAPK in AMs of CB rats could be described as follows: PTK-PI3K-Akt-JNK/p38 or PTK-PI3K-PKC-ERK, and PI3K may have a negative regulation on the activation of downstream proteins.

p53 is a key regulator for osthole-triggered cancer pathogenesis.

Osthole has been reported to have antitumor activities via the induction of apoptosis and inhibition of cancer cell growth and metastasis. However, the detailed molecular mechanisms underlying the anticancer effects of osthole in human colon cancer remain unclear. In the present study, we have assessed osthole-induced cell death in two different human colon cancer cell lines, HCT116 and SW480. Our results also showed that osthole activated proapoptotic signaling pathways in human colon cancer cells. By using cell culture insert system, osthole reduced cell motility in both human colon cancer cell lines. This study also provides evidence supporting the potential of osthole in p53 activation. Expression of p53, an apoptotic protein, was remarkably upregulated in cells treated with osthole. Importantly, the levels of phosphorylation of p53 on Ser15 (p-p53) and acetylation of p53 on Lys379 (acetyl-p53) were increased under osthole treatment. Our results also demonstrated that p53 was activated followed by generation of reactive oxygen species (ROS) and activation of c-Jun N-terminal kinase (JNK). Our study provides novel insights of p53-mediated responses under osthole treatment. Taken together, we concluded that osthole induces cancer cell death and inhibits migratory activity in a controlled manner and is a promising candidate for antitumor drug development.