Stress-activated Protein kinase/c-Jun N-terminal Kinase Research Articles

Oncostatin M enhances osteoprotegerin synthesis but reduces macrophage colony‑stimulating factor synthesis in bFGF‑stimulated osteoblast‑like cells.

Bone remodeling is tightly controlled by various factors, including hormones, autacoids and cytokines. Among them, oncostatin M (OSM) is a multifunctional cytokine produced by osteal macrophages, which serves as an essential modulator of bone remodeling. Macrophage colony-stimulating factor (M-CSF) and osteoprotegerin are secreted by osteoblasts, and also have pivotal roles in the regulation of the bone remodeling process. The binding of basic fibroblast growth factor (bFGF), a key regulator of bone remodeling, to the corresponding receptor [fibroblast growth factor receptor (FGFR)] triggers the dimerization and activation of FGFRs, which causes the phosphorylation of FGFR substrates and subsequent activation of downstream effectors, including mitogen-activated protein kinases (MAPKs), via Grb2. bFGF can activate MAPKs, resulting in the synthesis of osteoprotegerin and vascular endothelial growth factor in osteoblast-like MC3T3-E1 cells. In the present study, the effects of OSM on bFGF-induced osteoblast activation were investigated in the synthesis of osteoprotegerin and M-CSF in osteoblasts. The release of osteoprotegerin and M-CSF were analyzed using ELISA. The mRNA expression levels of osteoprotegerin and M-CSF were analyzed using reverse transcription-quantitative PCR. Phosphorylation of p38 MAPK, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p44/p42 MAPK was assessed using western blotting. OSM enhanced bFGF-induced osteoprotegerin release and bFGF-stimulated mRNA expression of osteoprotegerin. By contrast, OSM suppressed the bFGF-induced release of M-CSF and bFGF-stimulated mRNA expression of M-CSF. SB203580, a p38 MAPK inhibitor, and SP600125, a SAPK/JNK inhibitor, suppressed the bFGF-stimulated M-CSF release, whereas PD98059, an upstream kinase inhibitor of p44/p42 MAPK, failed to suppress the M-CSF release stimulated by bFGF. Furthermore, OSM enhanced the bFGF-induced phosphorylation of p38 MAPK, but attenuated the bFGF-stimulated phosphorylation of SAPK/JNK. By contrast, OSM had little effect on the bFGF-induced phosphorylation of p44/p42 MAPK. SB203580 markedly reduced the amplification of bFGF-stimulated osteoprotegerin release enhanced by OSM. These results strongly suggested that OSM may possess divergent effects on bFGF-induced osteoblast activation, upregulation of p38 MAPK and downregulation of SAPK/JNK, leading to the amplification of osteoprotegerin synthesis and the attenuation of M-CSF synthesis.

Oncostatin M stimulates prostaglandin D2-induced osteoprotegerin and interleukin-6 synthesis in osteoblasts

Oncostatin M produced by osteal macrophages plays a significant role in fracture healing. Osteoprotegerin (OPG) secreted by osteoblasts, binds to the receptor activator of nuclear factor-κB (RANK) ligand (RANKL) as a decoy receptor and prevents RANKL from binding to RANK, resulting in bone resorption suppression. Interleukin-6 (IL-6) is a pro-inflammatory cytokine and generally regulates bone resorption. However, accumulating evidence suggests that IL-6 plays pivotal roles in bone formation. We previously showed that prostaglandin D2 (PGD2) induces OPG synthesis by activating p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. Furthermore, we demonstrated that PGD2 stimulates IL-6 synthesis by activating p38 MAP kinase and p44/p42 MAP kinase in MC3T3-E1 cells. In the present study, we investigated whether oncostatin M affects PGD2-stimulated OPG and IL-6 synthesis in MC3T3-E1 cells through MAP kinase activation. The osteoblast-like MC3T3-E1 cells and normal human osteoblasts were treated with oncostatin M and subsequently stimulated with PGD2. Consequently, oncostatin M significantly increased the PGD2-stimulated OPG and IL-6 release in both cells. Oncostatin M significantly enhanced mRNA expression levels of OPG and IL-6 induced by PGD2 similarly in both cells. Regarding the signaling mechanism, oncostatin M did not affect the phosphorylation of p38 MAP kinase, SAPK/JNK, and p44/p42 MAP kinase. Our results suggest that oncostatin M upregulates the PGD2-stimulated OPG and IL-6 synthesis in osteoblasts and therefore affects bone remodeling. However, OPG and IL-6 synthesis are not mediated through p38 MAP kinase, p44/p42 MAP kinase, or SAPK/JNK pathways.

HSP70 inhibitors upregulate prostaglandin E1-induced synthesis of interleukin-6 in osteoblasts.

Interleukin-6 (IL-6) is a pro-inflammatory and bone-resorptive cytokine that also regulates bone formation. We previously showed that prostaglandin E1 (PGE1) induces the synthesis of IL-6 by activating p44/p42 mitogen-activated protein kinase (MAPK), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and p38 MAPK in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether heat shock protein 70 (HSP70), a molecular chaperone that coordinates protein folding and homeostasis, affects PGE1-stimulated IL-6 synthesis in MC3T3-E1 cells through the MAPK activation. The osteoblast-like MC3T3-E1 cells were treated with HSP70 inhibitors-VER-155008 and YM-08-, PD98059, SB203580 or SP600125 and then stimulated with PGE1. IL-6 synthesis was evaluated using an IL-6 enzyme-linked immunosorbent assay kit. IL-6 mRNA expression was measured by real-time RT-PCR. The phosphorylation of p38 MAPK was evaluated by Western blotting. We found that VER-155008, an HSP70 inhibitor, enhanced the PGE1-stimulated IL-6 release and IL-6 mRNA expression. YM-08, another HSP70 inhibitor, also enhanced PGE1-stimulated IL-6 release. PD98059, a p44/p42 MAPK inhibitor, and SP600125, a SAPK/JNK inhibitor, upregulated PGE1-stimulated IL-6 release. On the other hand, SB203580, a p38 MAPK inhibitor, suppressed PGE1-stimulated IL-6 release. YM-08 stimulated the PGE1-induced phosphorylation of p38 MAPK. SB203580 suppressed the amplification by YM-08 of the PGE1-stimulated IL-6 release. Our results suggest that HSP70 inhibitors upregulate the PGE1-stimulated IL-6 synthesis through p38 MAPK in osteoblasts and therefore affect bone remodeling.

Caffeic Acid Phenethyl Ester Induces Vascular Endothelial Growth Factor Production and Inhibits CXCL10 Production in Human Dental Pulp Cells.

The survival rate of root non-vital teeth is lower than that of vital teeth. Therefore, to preserve the dental pulp is very important. The vascular endothelial growth factor (VEGF) is the most potent angiogenic factor involved in the vitality of dental pulp including reparative dentin formation. Caffeic acid phenethyl ester (CAPE) is a physiologically active substance of propolis and has some bioactivities such as anti-inflammatory effects. However, there are no reports on the effects of CAPE on dental pulp inflammation. In this study, we investigated the effects of CAPE on VEGF and inflammatory cytokine production in human dental pulp cells (HDPCs) to apply CAPE to an ideal dental pulp protective agent. We found that CAPE induced VEGF production from HDPCs. Moreover, CAPE induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases (ERK), and stress-activated protein kinase/c-Jun N-terminal kinase (SAP/JNK) in HDPCs. Furthermore, CAPE inhibited C-X-C motif chemokine ligand 10 (CXCL10) production in Pam3CSK4- and tumor necrosis factor-alpha (TNF-α)-stimulated HDPCs. In conclusion, these results suggest that CAPE might be useful as a novel biological material for vital pulp therapy by exerting the effects of VEGF production and anti-inflammatory activities.

The Traditional Mongolian Medicine Qiqirigan-8 Effects on Lipid Metabolism and Inflammation in Obesity: Pharmacodynamic Evaluation and Relevant Metabolites.

Objective: Traditional Mongolian Medicine Qiqirigan-8 (MMQ-8) is a Chinese botanical drug with effective pharmacological properties in obesity. However, the pharmacological mechanism of MMQ-8 remains unclear. This study aimed to determine the active metabolites of MMQ-8 and its therapeutic effects on lipid metabolism and inflammation. Methods: The active metabolites of MMQ-8 were identified by ultrahigh-performance liquid chromatograph Q extractive mass spectrometry (UHPLC-QE-MS) assay and network analysis. An obesity rat model induced by high-fat diet was used in the study. Serum levels of lipids and inflammatory factors were detected using biochemical analysis and enzyme-linked immunosorbent assay (ELISA). Pathological analysis of liver tissues and arteries was conducted with hematoxylin and eosin (H&E) staining and immunohistochemistry. Protein expression of the tumor necrosis factor (TNF) signaling pathway was investigated by Western-blot. Simultaneously, bone marrow cells were used for RNA sequencing and relevant results were validated by cell culture and quantitative real-time polymerase chain reaction (RT-qPCR). Results: We identified 69 active metabolites and 551 target genes of MMQ-8. Of these, there are 65 active metabolites and 225 target genes closely related to obesity and inflammation. In vivo, we observed that MMQ-8 had general decreasing effects on body weight, white adipose tissue weight, and serum lipids. MMQ-8 treatment notably decreased the liver function markers and hepatic steatosis, and significantly decreased inflammation. In serum, it notably decreased TNF-α, interleukin (IL)-6, and inducible nitric oxide synthase (INOS), while elevating IL-10 levels. MMQ-8 treatment also significantly inhibited proteins phosphorylation of nuclear factor-kappa B inhibitor alpha (IκBα), mitogen-activated protein kinase (p38), extracellular regulated kinase 1/2(ERK1/2), and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and decreased vascular endothelium damage and macrophage infiltration and polarization to M1. These findings coincide with the RNA-sequencing data of bone marrow cells and results of in vitro experiments. Conclusion: We determined the pharmacological actions and relevant metabolites of MMQ-8 in obesity for the first time. Our study revealed MMQ-8 can optimize lipid metabolism and reduce chronic inflammation in obesity. However, more in-depth research is needed, for example, to understand the principle of compound compatibility and the inhibition effects on hepatic steatosis, T cell differentiation, and inflammatory signal transduction.

Upregulation of TGF-β-induced HSP27 by HSP90 inhibitors in osteoblasts

BackgroundHeat shock protein (HSP) 90 functions as a molecular chaperone and is constitutively expressed and induced in response to stress in many cell types. We have previously demonstrated that transforming growth factor-β (TGF-β), the most abundant cytokine in bone cells, induces the expression of HSP27 through Smad2, p44/p42 mitogen-activated protein kinase (MAPK), p38 MAPK, and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in mouse osteoblastic MC3T3-E1 cells. This study investigated the effects of HSP90 on the TGF-β-induced HSP27 expression and the underlying mechanism in mouse osteoblastic MC3T3-E1 cells.MethodsClonal osteoblastic MC3T3-E1 cells were treated with the HSP90 inhibitors and then stimulated with TGF-β. HSP27 expression and the phosphorylation of Smad2, p44/p42 MAPK, p38 MAPK, and SAPK/JNK were evaluated by western blot analysis.ResultHSP90 inhibitors 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) and onalespib significantly enhanced the TGF-β-induced HSP27 expression. TGF-β inhibitor SB431542 reduced the enhancement by 17-DMAG or onalespib of the TGF-β-induced HSP27 expression levels. HSP90 inhibitors, geldanamycin, onalespib, and 17-DMAG did not affect the TGF-β-stimulated phosphorylation of Smad2. Geldanamycin did not affect the TGF-β-stimulated phosphorylation of p44/p42 MAPK or p38 MAPK but significantly enhanced the TGF-β-stimulated phosphorylation of SAPK/JNK. Onalespib also increased the TGF-β-stimulated phosphorylation of SAPK/JNK. Furthermore, SP600125, a specific inhibitor for SAPK/JNK, significantly suppressed onalespib or geldanamycin’s enhancing effect of the TGF-β-induced HSP27 expression levels.ConclusionOur results strongly suggest that HSP90 inhibitors upregulated the TGF-β-induced HSP27 expression and that these effects of HSP90 inhibitors were mediated through SAPK/JNK pathway in osteoblasts.

Incretins Enhance PGF2α-Induced Synthesis of IL-6 and Osteoprotegerin in Osteoblasts.

Incretins including glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), which are secreted from the small intestine after oral food ingestion, are currently well-known to stimulate insulin secretion from pancreatic β-cells and used for the treatment of type 2 diabetes mellitus. We have previously reported that prostaglandin F2α (PGF2α) stimulates the synthesis of interleukin-6 (IL-6) and osteoprotegerin in osteoblast-like MC3T3-E1 cells, and that IL-6 and osteoprotegerin release are mediated through the p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase or stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) pathways. In the present study, we investigated the effects of incretins including GLP-1 and GIP, on the PGF2α-induced synthesis of IL-6 and osteoprotegerin and examined the detailed mechanism in osteoblast-like MC3T3-E1 cells. We found that GIP and GLP-1 significantly stimulated the PGF2α-induced synthesis of IL-6 in osteoblast-like MC3T3-E1 cells. In addition, GIP and GLP-1 significantly enhanced the PGF2α-induced mRNA expression levels of IL-6. On the other hand, GIP and GLP-1 markedly stimulated the PGF2α-induced synthesis of osteoprotegerin. However, the phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, or JNK induced by PGF2α was not affected by GIP or GLP-1. Therefore, these results strongly suggest that incretins enhance the PGF2α-induced synthesis of IL-6 and osteoprotegerin in osteoblast-like MC3T3-E1 cells. However, these syntheses are not mediated through p44/p42 MAP kinase, p38 MAP kinase, or JNK pathways.

Protective effects of 6-gingerol on 6-hydroxydopamine-induced apoptosis in PC12 cells through modulation of SAPK/JNK and survivin activation.

Due to many therapeutic effects, Ginger (Zingiber officinale) is the most widely used spice around the world, including in Iran. Due to its potent anti-inflammatory and antioxidant effects, ginger may protect against neurodegenerative disorders. Here, we investigated the effects of 6-gingerol (the main bioactive compound in ginger) on 6-hydroxydopamine (6-OHDA)-induced cell death in PC12 cells. Cell viability, cell apoptosis, and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and survivin expression were measured using resazurin, propidium iodide (PI) and flow cytometry, and western blot analysis. 6-OHDA (100 μM) reduced the cell viability, increased apoptosis, increased the active form of SAPK/JNK, and decreased survivin protein level in PC12 exposed cells in a dose and time-dependent manner. Pretreatment with 6-gingerol significantly increased the viability and reduced apoptosis (2.5 and 5 µM). Also, pretreatment with 6-gingerol at 2.5 and 5 µM increased survivin whereas, 6-gingerol at 2.5 µM reduced (P-SAPK/JNK):(SAPK/JNK) levels to a level near that of the related control. According to the results, 6-gingerol blocks 6-OHDA-induced cell damage by suppressing oxidative stress and anti-apoptotic activity. Thus, 6-gingerol may process beneficial protective effects in slowing the progression of Parkinson's disease.

Amplification by tramadol of PGD2-induced osteoprotegerin synthesis in osteoblasts: Involvement of μ-opioid receptor and 5-HT transporter

Tramadol, a weak μ-opioid receptor (MOR) agonist with inhibitory effects on the reuptake of serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine, is an effective analgesic to chronic pains. Osteoprotegerin produced by osteoblasts is essential for bone remodeling to suppress osteoclastic bone resorption. We previously reported that prostaglandin D2 (PGD2) induces osteoprotegerin synthesis whereby p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) are involved in osteoblast-like MC3T3-E1 cells. Herein, we investigated the mechanism underlying the effect of tramadol on the PGD2-induced osteoprotegerin synthesis in these cells. Tramadol enhanced the PGD2-induced release and mRNA expression of osteoprotegerin. Naloxone, a MOR antagonist, reduced the amplification by tramadol of the PGD2-stimulated osteoprotegerin release. Not the selective norepinephrine reuptake inhibitor reboxetine but the selective serotonin reuptake inhibitors fluvoxamine and sertraline upregulated the PGD2-induced osteoprotegerin release, which was further amplified by morphine. Tramadol enhanced PGD2-stimulated phosphorylation of p38 MAP kinase and SAPK/JNK, but not p44/p42 MAP kinase. Both SB203580 and SP600125 suppressed the tramadol effect to enhance the PGD2-stimulated osteoprotegerin release. Tramadol enhanced the PGE2-induced osteoprotegerin release as well as PGD2. These results suggest that tramadol amplifies the PGD2-induced osteoprotegerin synthesis at the upstream of p38 MAP kinase and SAPK/JNK in the involvement of both MOR and 5-HT transporter in osteoblasts.

Photothermal Killing of A549 Cells and Autophagy Induction by Bismuth Selenide Particles.

With a highly efficient optical absorption capability, bismuth selenide (Bi2Se3) can be used as an outstanding photothermal agent for anti-tumor treatment and shows promise in the field of nanotechnology-based biomedicine. However, little research has been completed on the relevant mechanism underlying the photothermal killing effect of Bi2Se3. Herein, the photothermal effects of Bi2Se3 particles on A549 cells were explored with emphasis put on autophagy. First, we characterized the structure and physicochemical property of the synthesized Bi2Se3 and confirmed their excellent photothermal conversion efficiency (35.72%), photostability, biocompatibility and ability of photothermal killing on A549 cells. Enhanced autophagy was detected in Bi2Se3-exposed cells under an 808 nm laser. Consistently, an elevated expression ratio of microtubule-associated protein 1 light chain 3-II (LC3-II) to LC3-I, a marker of autophagy occurrence, was induced in Bi2Se3-exposed cells upon near infrared (NIR) irradiation. Meanwhile, the expression of cleaved-PARP was increased in the irradiated cells dependently on the exposure concentrations of Bi2Se3 particles. Pharmacological inhibition of autophagy by 3-methyladenine (3-MA) further strengthened the photothermal killing effect of Bi2Se3. Meanwhile, stress-related signaling pathways, including p38 and stress activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), were activated, coupled with the attenuated PI3K/Akt signaling. Our study finds that autophagy and the activation of stress-related signaling pathways are involved in the photothermal killing of cancerous cells by Bi2Se3, which provides a more understanding of photothermal materials.

Attenuation by HSP90 inhibitors of EGF-elicited migration of osteoblasts: involvement of p44/p42 MAP kinase

ABSTRACT Background: We have demonstrated that epidermal growth factor (EGF)-induced migration of osteoblast-like MC3T3-E1 cells is mediated through p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase, stress-activated protein kinase/ c-Jun N-terminal kinase (SAPK/JNK), and Akt.The molecular chaperone heat shock protein 90 (HSP90) is abundantly expressed in osteoblasts. However, the role of HSP90 in osteoblast migration remains obscure. Objective: In this study, we investigated the effect of HSP90 inhibitors on the EGF-induced migration of MC3T3-E1 cells and the mechanism. Methods: Clonal osteoblast-like MC3T3-E1 cells were treated with the HSP90 inhibitors geldanamycin or onalespib and then stimulated with EGF. Cell migration was evaluated using the transwell cell migration assay and wound-healing assay. The viability of MC3T3-E1 cells was analyzed using the Cell Counting Kit-8. The phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, SAPK/JNK, Akt, and protein kinase-like endoplasmic reticulum kinase (PERK) was evaluated by western blot analysis. Results: EGF-induced migration was significantly suppressed by geldanamycin and onalespib, evaluated by both transwell cell migration assay and wound-healing assay. Geldanamycin and onalespib did not significantly alter cell viability. Geldanamycin and onalespib markedly reduced the EGF-induced phosphorylation of p44/p42 MAP kinase, but not p38 MAP kinase or Akt. By contrast, geldanamycin and onalespib increased the EGF-induced phosphorylation of SAPK/JNK. PERK phosphorylation was not significantly affected by geldanamycin or onalespib. Conclusion: Our results strongly suggest that HSP90 inhibitors reduce the EGF-induced osteoblast migration through the p44/p42 MAP kinase.

Acetaminophen reduces osteoprotegerin synthesis stimulated by PGE2 and PGF2α in osteoblasts: attenuation of SAPK/JNK but not p38 MAPK or p44/p42 MAPK.

Acetaminophen is one of the most widely used analgesic and antipyretic medicines, whose long-period use has reportedly been associated with an increased risk of bone fracture. However, the mechanism underlying this undesired effect remains to be investigated. The homeostatic control of bone tissue depends on the interaction between osteoblasts and osteoclasts. Osteoprotegerin produced by osteoblasts is known to play an essential role in suppressing osteoclast induction. We have previously reported that prostaglandin (PG) E2 and PGF2α induce osteoprotegerin synthesis through p38 mitogen-activated protein kinase (MAPK), p44/p42 MAPK and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effects of acetaminophen on the osteoprotegerin synthesis induced by PGE2 and PGF2α in MC3T3-E1 cells. Acetaminophen significantly suppressed the osteoprotegerin release stimulated by PGE2 and PGF2α. The PGE2-induced expression of osteoprotegerin mRNA was also reduced by acetaminophen. Acetaminophen markedly downregulated the phosphorylation of SAPK/JNK stimulated by PGE2 and PGF2α, but not those of p38 MAPK or p44/p42 MAPK. SP600125, an inhibitor of SAPK/JNK, suppressed the levels of PGE2- and PGF2α-upregulated osteoprotegerin mRNA expression. Taken together, these results strongly suggest that acetaminophen reduces the PGE2- and PGF2α-stimulated synthesis of osteoprotegerin in osteoblasts, and that the suppressive effect is exerted via attenuation of SAPK/JNK. These findings provide a molecular basis for the possible effect of acetaminophen on bone tissue metabolism.

GLP-1 reduces the migration of hepatocellular carcinoma cells via suppression of the stress-activated protein kinase/c-Jun N-terminal kinase pathway

Incretins, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), are hormones secreted from small intestine accompanied with oral intake. We previously showed that transforming growth factor (TGF)-α stimulates the migration of hepatocellular carcinoma (HCC) cells via mitogen-activated protein (MAP) kinases, AKT and Rho-kinase. However, it remains to be elucidated whether incretins affect HCC cell functions. In the present study, therefore, we investigated whether incretins affect the migration of HCC cells using human HCC-derived HuH7 cells. GLP-1, but not GIP, reduced both TGF-α- and hepatocyte growth factor (HGF)-induced cell migration. IBMX, an inhibitor of cyclic nucleotide phosphodiesterase, enhanced the suppressive effect of GLP-1. GLP-1 attenuated the phosphorylation of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) by TGF-α and HGF. Our results strongly suggest that GLP-1 suppresses TGF-α- and HGF-induced migration of HCC cells through inhibiting the SAPK/JNK signaling pathway, and that the inhibition by GLP-1 is due to cAMP production.

Mesenchymal stem cell carriers enhance antitumor efficacy induced by oncolytic reovirus in acute myeloid leukemia

Chemotherapy is the main treatment for acute myeloid leukemia (AML), but the therapeutic efficacy is modest, and most commonly manifests as relapse from remission. Thus, improving long-term AML survival is a crucial clinical challenge. In recent years, oncolytic virotherapy has provided an alternative approach for AML treatment. The use of oncolytic reoviruses has been explored in more than 30 clinical trials for safety and feasibility issues. However, like other oncolytic viruses, neutralizing antibodies (NAbs) reduce therapeutic efficacy. To tackle this problem, human umbilical cord mesenchymal stem cells (hUC-MSCs) were used to deliver reovirus using in vitro and in vivo models. Human UC-MSCs were successfully loaded with reovirus, without impairing biological function.We also observed in vitro protective effects of hUC-MSCs on reovirus in the presence of NAbs. In the immunocompromised AML mouse model, hUC-MSCs effectively carried reoviruses to tumor lesions and significantly prolonged the survival of AML xenografts in mice in the presence of a high titer anti-reovirus antibody (p = 0.001). However, reovirus-induced activation of AKT, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and NF-κB signaling led to the maintenance of intrinsic migratory properties and secretion of pro-inflammatory cytokines from hUC-MSCs, particularly CXCL10. In immuno-competent AML mice, MSCs carrying reovirus triggered immune responses, and eventually inhibited tumor growth. Therefore, these results suggest that MSCs as carriers of oncolytic reoviruses can enhance the antitumor efficacy of virotherapy.

Tramadol enhances PGF2α-stimulated osteoprotegerin synthesis in osteoblasts

Osteoprotegerin (OPG) synthesized by osteoblasts is currently considered a crucial regulator to suppress the formation and function of osteoclasts. We previously showed that the synthesis of OPG is stimulated by prostaglandin F2α (PGF2α) in the involvement of p38 mitogen-activated protein kinase (MAPK), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p44/p42 MAPK in osteoblast-like MC3T3-E1 cells. We also found that Rho-kinase is involved in the signaling of PGF2α upstream of p38 MAPK in these cells. Tramadol is widely used to treat chronic pain, such as low back pain associated with osteoporosis. We investigated whether or not tramadol affects the OPG release induced by PGF2α in osteoblast-like MC3T3-E1 cells. The levels of OPG in the conditioned medium were measured by an enzyme-linked immunosorbent assay. The mRNA expression of OPG was determined with real-time reverse transcription polymerase chain reaction. The phosphorylation of target protein was determined with a Western blot analysis. PGF2α induced the release and the mRNA expression of OPG, which tramadol significantly enhanced. Morphine, a selective μ-opioid receptor (MOR) agonist, also enhanced the PGF2α-induced OPG release. In addition, naloxone, a MOR antagonist, suppressed the enhancement by tramadol or morphine of the PGF2α-induced OPG synthesis. Tramadol upregulated the phosphorylation of SAPK/JNK and p38 MAPK stimulated by PGF2α but not that of p44/p42 MAPK or myosin phosphatase targeting protein (MYPT), a substrate of Rho-kinase. The inhibitors of both p38 MAPK and SAPK/JNK, SB203580 and SP600125, respectively, reduced the tramadol amplification of OPG release stimulated by PGF2α. The present results strongly suggest that tramadol enhances the synthesis of OPG stimulated by PGF2α through MOR in osteoblasts, and that the amplifying effect is exerted at upstream of p38 MAPK and SAPK/JNK but downstream of Rho-kinase.

Indirubin 3′-Oxime Inhibits Migration, Invasion, and Metastasis InVivo in Mice Bearing Spontaneously Occurring Pancreatic Cancer via Blocking the RAF/ERK, AKT, and SAPK/JNK Pathways

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with high invasive and metastatic potential. We generated a spontaneous PDAC mouse model and examined the therapeutic potential of indirubin 3′-oxime (Indox) against PDAC bearing mouse in vivo. METHODS: Randomized 3-month-old LSL-KrasG12D/+;Trp53flox/+;Pdx-1-cre (KPCflox) mice were intraperitoneally injected with 40 mg/kg Indox (n = 9) or a vehicle (n = 10) twice a week. At the end point, tumor status including proliferation, direct invasion, and distant metastasis was analyzed histopathologically. The inhibitory potentials of Indox for proliferation, migration/invasion, and the phosphorylation of target molecules were determined in KPCflox-derived PDAC cells in vitro. RESULTS: Prolonged survival by Indox via intraperitoneal administration was observed in the KPCflox mice. Indox inhibited tumor proliferation accompanied with low levels of nuclear phosphorylated cyclin-dependent kinase (p-CDK) and cyclin B1 in vivo. Furthermore, Indox inhibited the migration/invasive activities of PDAC via down-regulation of matrix metalloproteinase (MMP)-9 in vitro and in vivo. Antibody array and immunoblotting analysis revealed that Indox inhibited the phosphorylation of multiple molecules, including key upstream proteins of MMP-9 in RAF/extracellular signal-regulated kinase (ERK), AKT, and stress-activated protein kinase/c-Jun-N-terminal kinase (SAPK/JNK) pathways. CONCLUSION: Indox inhibited the proliferative, invasive, and metastatic potentials of PDAC in vitro and in vivo. Therefore, Indox could a therapeutic candidate for treating spontaneously occurring PDAC via blocking the RAF/ERK, AKT and SAPK/JNK pathways.

Olive oil polyphenols suppress the TGF-α-induced migration of hepatocellular carcinoma cells.

Oleuropein and 3-hydroxytyrosol (3-HT) are natural polyphenols present in olive oil that are known to exhibit potent anti-oxidant activities and exert protective effects against a number of human diseases. In the liver, olive oil polyphenols have been demonstrated to prevent hepatocellular carcinoma (HCC) cell growth. However, little is known about their effects against HCC cell migration. Therefore, the present study investigated whether or not oleuropein and 3-HT were involved in the suppression of transforming growth factor-α (TGF-α)-induced migration of human HCC cells using human HCC-derived HuH7 cells. The TGF-α-induced migration of HuH7 cells was significantly and dose-dependently suppressed by oleuropein and 3-HT. This study group demonstrated previously that the TGF-α-induced activation of AKT and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) were involved in HuH7 cell migration. In addition to these protein kinases, the present study examined the involvement of TGF-α-induced activation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and Rho kinase in HuH7 cell migration. TGF-α-induced HuH7 cell migration was decreased by SB203580, a p38 MAPK inhibitor, and Y27632, a Rho kinase inhibitor. However, PD98059, an inhibitor of the upstream kinase activating ERK, did not suppress the TGF-α-induced migration of HuH7 cells. Although AKT, SAPK/JNK, p38 MAPK and Rho kinase pathways were suggested to be involved in the TGF-α-induced migration of HuH7 cells, 10-30 µM 3-HT did not exhibit any suppressive effect on the TGF-α-stimulated activities of these kinases. The results of the present study suggest that olive oil polyphenols suppressed the TGF-α-induced migration of HCC cells.

Enhancement by HSP90 inhibitor of PGD2-stimulated HSP27 induction in osteoblasts: Suppression of SAPK/JNK and p38 MAP kinase

Heat shock protein (HSP) 90 that is ubiquitously expressed in various tissues is a major molecular chaperone. We have previously demonstrated that prostaglandin D2 (PGD2), a bone remodeling factor, elicits the expression of HSP27, a small HSP, through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of HSP90 in the PGD2-stimulated HSP27 induction and the underlying mechanism in MC3T3-E1 cells. Onalespib, an inhibitor of HSP90, significantly enhanced the PGD2-stimulated HSP27 induction. In addition, geldanamycin, another HSP90 inhibitor, potentiated the HSP27 induction. Both onalespib and geldanamycin markedly amplified the PGD2-induced phosphorylation of SAPK/JNK and p38 MAP kinase. SP600125, an inhibitor of SAPK/JNK, and SB203580, an inhibitor of p38 MAP kinase, suppressed the amplification by onalespib of the PGD2-stimulated HSP27 induction. These results strongly suggest that HSP90 plays a negative role in the HSP27 induction stimulated by PGD2 in osteoblasts, and that the inhibitory effect of HSP90 is mediated through the regulation of SAPK/JNK and p38 MAP kinase.

HSP70 Inhibitors Reduce The Osteoblast Migration By Epidermal Growth Factor.

We have recently reported that epidermal growth factor (EGF) induces migration of osteoblast-like MC3T3-E1 cells through the activation of p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase, stress-activated protein kinase/ c-Jun N-terminal kinase (SAPK/JNK) and Akt. Furthermore, we demonstrated that heat shock protein 70 (HSP70) down-regulates the transforming growth factor-β- stimulated vascular endothelial growth factor synthesis via suppression of p38 MAP kinase in osteoblast-like MC3T3-E1 cells. However, the exact role of HSP70 underlying osteoblast migration is not fully elucidated. The aim of this study is to investigate the effects of HSP70 inhibitors on the EGF-stimulated osteoblast migration, and the underlying mechanism. Osteoblast-like MC3T3-E1 cells were treated with two types of HSP70 inhibitors, VER-155008 or YM-08. Transwell cell migration assay and wound-healing assay were analyzed for osteoblast migration. The expression levels of HSP70 and the phosphorylation of p38 MAP kinase, p44/p42 MAP kinase, SAPK/JNK or Akt were evaluated by a Western blot analysis. EGF hardly affected the expression levels of HSP70 at the present or absent of VER-155008. EGF-stimulated migration was significantly reduced by both HSP70 inhibitors, VER-155008 and YM-08, determined by a transwell cell migration assay. The suppressive effects of both HSP70 inhibitors on the migration stimulated by EGF were also observed by a wound-healing assay. VER-155008 inhibited the EGF-induced phosphorylation of p44/p42 MAP kinase and AKT, but not p38 MAP kinase or SAPK/JNK. This study provides new evidence that HSP70 inhibitors reduce the EGFstimulated migration of osteoblasts through the suppression of p44/p42 MAP kinase and Akt.

FABP7 Protects Astrocytes Against ROS Toxicity via Lipid Droplet Formation.

Fatty acid-binding proteins (FABPs) bind and internalize long-chain fatty acids, controlling lipid dynamics. Recent studies have proposed the involvement of FABPs, particularly FABP7, in lipid droplet (LD) formation in glioma, but the physiological significance of LDs is poorly understood. In this study, we sought to examine the role of FABP7 in primary mouse astrocytes, focusing on its protective effect against reactive oxygen species (ROS) stress. In FABP7 knockout (KO) astrocytes, ROS induction significantly decreased LD accumulation, elevated ROS toxicity, and impaired thioredoxin (TRX) but not peroxiredoxin 1 (PRX1) signalling compared to ROS induction in wild-type astrocytes. Consequently, activation of apoptosis signalling molecules, including p38 mitogen-activated protein kinase (MAPK) and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and increased expression of cleaved caspase 3 were observed in FABP7 KO astrocytes under ROS stress. N-acetyl L-cysteine (NAC) application successfully rescued the ROS toxicity in FABP7 KO astrocytes. Furthermore, FABP7 overexpression in U87 human glioma cell line revealed higher LD accumulation and higher antioxidant defence enzyme (TRX, TRX reductase 1 [TRXRD1]) expression than mock transfection and protected against apoptosis signalling (p38 MAPK, SAPK/JNK and cleaved caspase 3) activation. Taken together, these data suggest that FABP7 protects astrocytes from ROS toxicity through LD formation, providing new insights linking FABP7, lipid homeostasis, and neuropsychiatric/neurodegenerative disorders, including Alzheimer's disease and schizophrenia.