Inhibition Of JNK Activation Research Articles

JNK in spinal cord facilitates bone cancer pain in rats through modulation of CXCL1.

In patients with advanced cancer, cancer-induced bone pain (CIBP) is a severe and common problem that is difficult to manage and explain. As c-Jun N-terminal kinase (JNK) and chemokine (C-X-C motif) ligand 1 (CXCL1) have been shown to participate in several chronic pain processes, we investigated the role of JNK and CXCL1 in CIBP and the relationship between them. A rat bone cancer pain model was established by intramedullary injection of Walker 256 rat gland mammary carcinoma cells into the left tibia of Sprague-Dawley rats. As a result, intramedullary injection of Walker 256 carcinoma cells induced significant bone destruction and persistent pain. Both phosphorylated JNK1 (pJNK1) and pJNK2 showed time-dependent increases in the ipsilateral spinal cord from day 7 to day 18 after tumor injection. Inhibition of JNK activation by intrathecal administration of SP600125, a selective pJNK inhibitor, attenuated mechanical allodynia and heat hyperalgesia caused by tumor inoculation. Tumor cell inoculation also induced robust CXCL1 upregulation in the ipsilateral spinal cord on day 18 after tumor injection. Inhibition of CXCL1 by intrathecal administration of CXCL1 neutralizing antibody showed a stable analgesic effect. Intrathecal administration of SP600125 reduced CXCL1 increase in the spinal cord, whereas inhibition of CXCL1 in the spinal cord showed no influence on JNK activation. Taken together, these results suggested that JNK activation in spinal cord contributed to the maintenance of CIBP, which may act through modulation of CXCL1. Inhibition of the pJNK/CXCL1 pathway may provide a new choice for treatment of CIBP.

Effects of a Sublethal and Transient Stress of the Endoplasmic Reticulum on the Mitochondrial Population.

Endoplasmic reticulum (ER) and mitochondria are not discrete intracellular organelles but establish close physical and functional interactions involved in several biological processes including mitochondrial bioenergetics, calcium homeostasis, lipid synthesis, and the regulation of apoptotic cell death pathways. As many cell types might face a transient and sublethal ER stress during their lifetime, it is thus likely that the adaptive UPR response might affect the mitochondrial population. The aim of this work was to study the putative effects of a non-lethal and transient endoplasmic reticulum stress on the mitochondrial population in HepG2 cells. The results show that thapsigargin and brefeldin A, used to induce a transient and sublethal ER stress, rapidly lead to the fragmentation of the mitochondrial network associated with a decrease in mitochondrial membrane potential, O2 (•-) production and less efficient respiration. These changes in mitochondrial function are transient and preceded by the phosphorylation of JNK. Inhibition of JNK activation by SP600125 prevents the decrease in O2 (•-) production and the mitochondrial network fragmentation observed in cells exposed to the ER stress but has no impact on the reduction of the mitochondrial membrane potential. In conclusion, our data show that a non-lethal and transient ER stress triggers a rapid activation of JNK without inducing apoptosis, leading to the fragmentation of the mitochondrial network and a reduction of O2 (•-) production. J. Cell. Physiol. 231: 1913-1931, 2016. © 2015 Wiley Periodicals, Inc.

Protective effect of apoptosis signal-regulating kinase 1 inhibitor against mice liver injury.

To explore the protective effect and its molecular mechanism of apoptosis signal-regulating kinase 1 (ASK1) inhibitor (GS-459679) on acetaminophen-induced liver injury in mice. The model of liver injury was established by administration of acetaminophen (APAP) (300mg/kg, i.p.) on C57BL/6 mice. Forty-eight male C57BL/6 mice were randomly divided into four groups, consisting of control group, GS group (GS-459679, 30mg/kg, i.p.), APAP-induced group, and GS combined with APAP-induced group. For GS combined with APAP-induced group, mice were treated with GS 30min prior to administration of APAP. After mice were euthanized at 6h or 12h, respectively, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed, and mRNA levels of TNF-α, IL-6 and IL-1β were tested. The activity of glutathione (GSH), oxidized GSH (GSSG) and malondialdehyde were quantified. In addition, ASK1, P-ASK1, JNK and P-JNK protein levels were tested in all groups. The ASK1 and P-ASK1 levels were up-regulated in APAP-induced group. Compared to the control group, serum levels of ALT and AST, and mRNA levels of TNF-α, IL-6 and IL-1β were increased in APAP-induced group. Meanwhile, the levels of MAD and GSSG, and the ratio of GSSG/GSH were higher and the JNK was activatedin APAP-induced group compared with that in control group. However, compared to APAP-induced group, GS combined with APAP-induced group displayed a decrease of protein expression levels of ASK1, P-ASK1 and P-JNK, a reduction of serum levels of ALT and AST, a decrease in TNF-α, IL-6 and IL-1β mRNA levels, and a low ration of GSSG/GSH. GS-459679 treatment effectively down-regulates ASK1 and P-ASK1 expression. Addition of GS-459679 decreases the generation of liver metabolites and inflammatory factors, reduces oxidative stress reaction, inhibits JNK activation, and then protects the responsiveness to APAP-induced liver injury.

CAMP signaling increases histone deacetylase 8 expression by inhibiting JNK-dependent degradation via autophagy and the proteasome system in H1299 lung cancer cells

This study aimed to investigate the roles of autophagy and the ubiquitin-proteasome system in the degradation of histone deacetylase 8 (HDAC8) and to clarify the mechanism by which cAMP signaling regulates this degradation. cAMP signaling was activated by treating H1299 non-small cell lung cancer cells with isoproterenol or forskolin/3-isobutyl-1-methylxanthine, and HDAC8 expression was assessed by western blot analysis. The inhibition of autophagy and ubiquitin-proteasome-dependent degradation increased HDAC8 expression. cAMP signaling inhibited JNK activation, which decreased the phosphorylation of Bcl-2, thereby reducing autophagy, and the phosphorylation of Itch, thereby reducing ubiquitination. These results suggest that the HDAC8 protein is degraded via autophagy and the ubiquitin-proteasome system and that cAMP signaling increases HDAC8 protein levels by reducing JNK-mediated autophagy and ubiquitin-proteasome-dependent degradation of the HDAC8 protein in H1299 lung cancer cells.

Deletion of β-Arrestin2 in Mice Limited Pancreatic β-Cell Expansion under Metabolic Stress through Activation of the JNK Pathway.

β-Arrestin2 (βarr2) is an adaptor protein that interacts with numerous signaling molecules and regulates insulin sensitivity. We reported previously that βarr2 was abundantly expressed in mouse pancreatic β-cells, and loss of βarr2 leads to impairment of acute- and late-phase insulin secretion. In the present study, we examined the dynamic changes of β-cell mass in βarr2-deficient (βarr2-/-) mice in vivo and explored the underlying mechanisms involved. βarr2-/- mice with exclusively luciferase overexpression in β-cells were generated and fed a high-fat diet (HFD). β-Cell mass was determined by in vivo noninvasive bioluminescence imaging from 4 to 20 wks of age. Proliferation was measured by 5-bromo-2-deoxyuridine (BrdU) incorporation and fluorescence-activated cell sorter analysis. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunoblotting were conducted for gene and protein expression. We found that β-cell mass was reduced dramatically in βarr2-/- mice at 12 wks old compared with that of their respective HFD-fed controls. The percentage of BrdU- and Ki67-positive cells reduced in islets from βarr2-/- mice. Exposure of βarr2-/- islets to high levels of glucose and free fatty acids (FFAs) exacerbated cell death, which was associated with upregulation of the JNK pathway in these islets. Conversely, overexpression of βarr2 amplified β-cell proliferation with a concomitant increase in cyclinD2 expression and a decrease in p21 expression and protected β-cells from glucose- and FFA-induced cell death through JNK-activation inhibition. In conclusion, βarr2 plays roles in regulation of pancreatic β-cell mass through the modulation of cell cycle regulatory genes and the inhibition of JNK activation induced by glucolipotoxity, which implicates a role for βarr2 in the development of type 2 diabetes.

(Z)3,4,5,4'-trans-tetramethoxystilbene, a new analogue of resveratrol, inhibits gefitinb-resistant non-small cell lung cancer via selectively elevating intracellular calcium level.

Calcium is a second messenger which is required for regulation of many cellular processes. However, excessive elevation or prolonged activation of calcium signaling would lead to cell death. As such, selectively regulating calcium signaling could be an alternative approach for anti-cancer therapy. Recently, we have identified an effective analogue of resveratrol, (Z)3,4,5,4′-trans-tetramethoxystilbene (TMS) which selectively elevated the intracellular calcium level in gefitinib-resistant (G-R) non-small-cell lung cancer (NSCLC) cells. TMS exhibited significant inhibitory effect on G-R NSCLC cells, but not other NSCLC cells and normal lung epithelial cells. The phosphorylation and activation of EGFR were inhibited by TMS in G-R cells. TMS induced caspase-independent apoptosis and autophagy by directly binding to SERCA and causing endoplasmic reticulum (ER) stress and AMPK activation. Proteomics analysis also further confirmed that mTOR pathway, which is the downstream of AMPK, was significantly suppressed by TMS. JNK, the cross-linker of ER stress and mTOR pathway was significantly activated by TMS. In addition, the inhibition of JNK activation can partially block the effect of TMS. Taken together, TMS showed promising anti-cancer activity by mediating calcium signaling pathway and inducing apoptosis as well as autophagy in G-R NSCLC cells, providing strategy in designing multi-targeting drug for treating G-R patients.

Lipid deposition in liver cells: The influence of short form augmenter of liver regeneration

The short form augmenter of liver regeneration (sfALR) is a novel human hepatotrophic growth factor. The aim of this study was to investigate the potential role of sfALR in NAFLD. The free fatty acids (FFA) induced lipid accumulation in mouse liver parenchymal cells was examined by Oil Red O staining and triglyceride level determination. The cell cycle was determined by flow cytometry and the proliferation was assessed by CCK8. The expression levels of gfer, miR-122, srebp-1c, fas, dgat2, acc1 and Lrp1B were assessed by quantitative real-time PCR. Furthermore, the MAPK pathway was detected by western blot. The results showed that sfALR could alleviate the lipid accumulation in mice both in vivo and in vitro. sfALR relieved the proliferation inhibition and G2 arrest of mouse liver parenchymal cells induced by FFAs. Free fatty acids affected gfer expression in a time-and dose-dependent way. And sfALR suppressed JNK activation, increased miR-122 level and reduced fatty acid synthesis-related gene expression. These findings suggested that sfALR could alleviate the severity of fatty liver in mice.

Abstract A56: Metformin targets Rho GTPases to inhibit neuroblastoma cell growth; implications in the treatment of neuroblastoma

Abstract In recent years, oncologists are paying considerable attention to metformin (N', N'-dimethylbiguanide) in the treatment of various types of cancers, as some population-based studies have shown low cancer incidences and mortalities among diabetic-patients treated with metformin. In the present study, using neuroblastoma cells, we explored the anti-tumor activity and the underlying mechanisms of metformin. Neuroblastoma is a malignant cancer of the postganglionic sympathetic nervous system; it develops in the adrenal gland and metastasizes to liver, bone, bone marrow, lymph nodes, neck and chest. It is the most common cancer in infants younger than one and second most common tumors in children. In the United States of America, it accounts for 7% of all childhood cancers, and is responsible for 15% of all cancer deaths in children younger than 15 years. Various genetic and cytological alterations allow cells to develop drug resistance and help neuroblastoma tumors to escape most available therapies. Using human neuroblastoma cells of different genotypes (SH-SY5Y, MYCN-nonamplified; and SK-N-BE(2), MYCN-amplified cells), we generated subcutaneous xenograft mice models, and metformin (50, 100 and 250 mg/kg body weight per mice) was given daily by oral gavage to tumor-bearing mice. Our in vivo results demonstrated that metformin (100 and 250 mg/kg body weight) significantly inhibited the growth of tumors in both mice models. Immunofluorescence and Western blot results indicated reduction in tumor growth was due to the increased DNA fragmentation and apoptotic cell death that occur via activation of caspase-3. Metformin also inhibited the initiation of spheroid formation in 3-D cultures (hanging drop assay). In xenograft tumors, metformin affected the phosphorylation of mitogen-activated protein kinases (MAPKs). Metformin induced the phosphorylation of JNK1/2 and inhibited the phosphorylation of ERK1/2 without affecting p38 MAP Kinase. Since ERK1/2 is involved in cell proliferation and JNK1/2 promotes cell death, our observations clearly indicated that MAP kinases are involved in metformin-induced cell death. Rho-GTPases (RhoA, Rac1 and Cdc42), a family of small G-proteins, act as molecular switches to regulate cellular functions- cell division, motility and cell survival. These small G-proteins exist either as a GTP-bound active form or an inactive GDP-bound form. The aberrant expression and/or activity of Rho-GTPases are associated with the progression of various cancers. Our glutathione-S-transferase (GST)-pull down assays showed that metformin significantly increased activation of Rac1 (GTP-Rac1) and Cdc42 (GTP-Cdc42) while it decreased activation of RhoA (GTP-RhoA) in these tumors. Infection of neuroblastoma cells by adenoviruses expressing dominant negative Rac1, Cdc42 (Rac1-N17 and Cdc42-N17, respectively) and constitutively active RhoA (RhoA-V14), or incubation with pharmacological inhibitors of Rac1 (NSC23766) or Cdc42 (ML141) significantly protected cells from metformin-induced apoptosis. Additionally, inhibition of JNK activity with Rac1 or Cdc42 attenuated cytotoxic effects of metformin. These gain-in and loss-off function studies suggested that metformin impairs Rho-GTPase signaling to initiate apoptosis via JNK pathway. Together, our results reported the inhibitory effects of metformin against neuroblastoma, and demonstrated the role of Rho-GTPases in metformin-mediated apoptotic cell death. The cytotoxic effects of metformin against MYCN-nonamplified and MYCN-amplified multidrug resistant neuroblastoma cells in our studies further signifies that metformin can be a promising drug candidate for neuroblastoma therapy. Citation Format: Ambrish Kumar, Nadia Al-Sammarraie, Donald J. DiPette, Ugra S. Singh. Metformin targets Rho GTPases to inhibit neuroblastoma cell growth; implications in the treatment of neuroblastoma. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr A56.

Abstract 2525: MLN4924 synergistically enhances cisplatin-induced cytotoxicity via JNK and Bcl-xL pathways in human urothelial carcinoma - In vitro and in vivo study

Abstract Introduction Cisplatin-based chemotherapy is the main treatment for metastatic bladder urothelial carcinoma; however, the response rate is only approximately 40-65%. Many patients will encounter drug resistance to chemotherapy eventually. Therefore, combination therapies to enhance the efficacy of cisplatin and to overcome drug resistance are imperative for the treatment of urothelial carcinoma. The aim of this study was to investigate the antitumor effect and underlying mechanisms for the combination of cisplatin and MLN4924, a NEDD8-activating enzyme inhibitor, in human bladder urothelial carcinoma. Materials & Methods We used MTT assays and CalcuSyn software to calculate the combination index of cisplatin and MLN4924. The expression levels of DNA damage response regulators, apoptotic-related molecules, MAPK and Bcl-xL were revealed by western blot. c-Jun N-terminal kinase (JNK) inhibitor (SP600125) and overexpression of Bcl-xL were used to investigate the relationship between JNK and Bcl-xL. The in vitro findings were further confirmed in vivo via xenograft mouse model. Results The combination of cisplatin and MLN4924 exerted synergistic cytotoxicity on two high-grade bladder urothelial carcinoma cell lines, NTUB1 and T24 (combination index <1). In addition, MLN4924 potentiated cisplatin-induced apoptosis and activation of caspase-3, 7, phospho-histone H2A.X and PARP. We also observed c-Jun N-terminal kinase (JNK) activation with down-regulation of Bcl-xL during cisplatin and MLN4924 treatment. The inhibition of JNK activation partially restored cell viability as well as the expression of B-cell lymphoma-extra large (Bcl-xL); moreover, the overexpression of Bcl-xL rescued cell viability. In the in vivo study, MLN4924 significantly potentiated cisplatin-induced tumor suppression in urothelial carcinoma xenograft mice. Conclusion In summary, MLN4924 synergistically enhanced the antitumor effect of cisplatin through increasing DNA damage, JNK activation and down-regulating Bcl-xL in urothelial carcinoma cells. These findings provide a new therapeutiuc strategy for bladder cancer. Citation Format: I-Lin Ho, Kuan-Lin Kuo, Chien-Tso Chou, Chen-Hsun Hsu, Yeong-Shiau Pu, Ju-Ton Hsieh, Kuo-How Huang. MLN4924 synergistically enhances cisplatin-induced cytotoxicity via JNK and Bcl-xL pathways in human urothelial carcinoma - In vitro and in vivo study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2525. doi:10.1158/1538-7445.AM2015-2525

Abstract 2660: Synergistic effect of PI3Kδ inhibitor CAL-101 and JNK inhibitor SP600125 on glioblastoma cell proliferation

Abstract Glioblastoma multiform (GBM) is the most aggressive malignant primary tumor in the central nervous system. Due to the hyperactivation of phosphatidylinositol 3-kinase (PI3K) and c-Jun N-terminal kinase (JNK) pathways in GBM, their respective inhibitors become promising and actively researched therapeutic agents. Our previous study showed that the PI3K catalytic isoform p110δ is highly expressed in GBM cell lines, indicating that p110δ may be a potential target for therapy. However, a single drug may not be sufficient to affect all pathogenic processes such as proliferation, migration and invasion in GBM cells. Therefore, we aimed to evaluate the combination effect of PI3Kδ inhibitor CAL-101 and JNK inhibitor SP600125 on glioma cells. The half maximal inhibitory concentration (IC50) of CAL-101 and SP600125 was first determined by the MTT method in GBM cell line U-87 MG. The combination effect of these 2 inhibitors on cell proliferation was analyzed using Compusyn software and the combination index (CI) was calculated. CI <0.9 indicates a synergistic effect, whereas CI >1.1 indicates an antagonistic effect, and CI between 0.9 and 1.1 indicates an additive effect. Cell migration and invasion capacity of U-87 MG cells were then evaluated under combined treatment with CAL-101 and SP600125. Our results showed that the effect of CAL-101 or SP600125 on cell proliferation was dose- and time-dependent. The IC50 of CAL-101 and SP600125 were 10.15 μM and 19.60 μM respectively. Treatment with SP600125 alone significantly suppressed U-87 MG cell proliferation, and combining 10 μM of CAL-101 and 20 μM of SP600125 showed a moderate synergistic effect. The cytotoxicity of CAL-101 or SP600125 alone, as well as combining them together was low on normal astrocytes. Western blot analysis of the PI3K/Akt and JNK pathways indicated that CAL-101 inhibited Akt phosphorylation at Ser473 and Thr308, whereas SP600125 suppressed JNK activity and Akt phosphorylation at Thr308. Combination of CAL-101 and SP600125 further inhibited Akt phosphorylation at Thr308, but showed no effect on the inhibition of Akt phosphorylation at Ser473 and c-Jun phosphorylation. CAL-101 significantly reduced migration but had no effect on invasion of U-87 MG cells, whereas SP600125 significantly suppressed both their migration and invasion abilities. Combining CAL-101 and SP600125 augmented the inhibitory effect on migration but showed no enhancement on invasion. In conclusion, combination treatment with CAL-101 and SP600125 showed a moderate synergistic effect on GBM cell proliferation, and enhanced the inhibition of cell migration. Our results suggest that GBM patients may benefit from combination therapy targeting both PI3Kδ and JNK. Citation Format: Hua-Fu ZHAO, Jing WANG, Shing-Shun Tony TO. Synergistic effect of PI3Kδ inhibitor CAL-101 and JNK inhibitor SP600125 on glioblastoma cell proliferation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2660. doi:10.1158/1538-7445.AM2015-2660

Formoterol synergy with des-ciclesonide inhibits IL-4 expression in IgE/antigen-induced mast cells by inhibiting JNK activation

Inhaled corticosteroid (ICS) therapy in combination with long-acting β-adrenergic agonists (LABA) is the most important treatment for allergic asthma, although the mechanism still remains unclear. However, mast cells play a central role in the pathogenesis of asthma. In this study, we explored the sole or synergetic effects of des-ciclesonide (ICS) and formoterol (LABA) on the cytokines IL-4 and IL-13 and on histamine release from mast cells (RBL-2H3 cells). We found that des-ciclesonide (0.1, 1 and 10nM) and formoterol (0.1, 1 and 10μM) alone attenuated DNP–BSA-induced IL-4 and IL-13 production, respectively, in a concentration-dependent manner in DNP-IgE-sensitized mast cells. Des-ciclesonide (0.2nM) and formoterol (1μM) alone also reduced histamine production. However, the combination of des-ciclesonide (0.2nM) and formoterol (1μM) had a synergistic inhibition effect on IL-4 mRNA expression and protein production but not IL-13 and histamine release. The JNK inhibitor SP600125 (10μM) inhibited antigen-induced mRNA expression and protein production of IL-4. Des-ciclesonide and formoterol alone inhibited the activation of JNK in a concentration-dependent manner, and the combination of des-ciclesonide (0.2nM) and formoterol (1μM) exhibited greater inhibition effect compared with des-ciclesonide (0.2nM) or formoterol (1μM) alone. Taken together, these synergistic effects on mast cells might provide the rationale for the development of the most recent ICS/LABA combination approved for asthma therapy.

Activation of the Liver X Receptor by Agonist TO901317 Improves Hepatic Insulin Resistance via Suppressing Reactive Oxygen Species and JNK Pathway.

Activation of Liver X receptors (LXRs), key transcriptional regulators of glucose metabolism, normalizes glycemia and improves insulin sensitivity in rodent models with insulin resistance. However, the molecular mechanism is unclear. This study is aimed to elucidate the mechanism of LXRs-mediated liver glucose metabolic regulation in vitro and in vivo. Db/db mice were used as an in vivo model of diabetes; palmitate (PA)-stimulated HepG2 cells were used as an in vitro cell model with impairment of insulin signaling. TO901317 (TO) was chosen as the LXRs agonist. We demonstrated that TO treatment for 14 days potently improved the hepatic glucose metabolism in db/db mice, including fasting blood glucose, fasting insulin level, and HOMA-IR. TO had no effect on the glucose metabolism in normal WT mice. TO-mediated activation of hepatic LXRs led to strong inhibition of ROS production accompanied by inactivation of JNK pathway and re-activation of Akt pathway. TO also suppressed the expression of gluconeogenic genes such as PEPCK and G-6-pase in db/db mice, but not in WT mice. In HepG2 cells, TO almost completely restored PA-induced Akt inactivation, and suppressed PA-stimulated ROS production and JNK activation. Interestingly, basal level of ROS was also inhibited by TO in HepG2 cells. TO significantly inhibited PA-stimulated expressions of gluconeogenic genes. Finally, we found that anti-oxidative genes, such as Nrf2, were up-regulated after LXRs activation by TO. These results strongly support the notion that activation of LXRs is critical in suppression of liver gluconeogenesis and improvement of insulin sensitivity in diabetic individuals. At molecular levels, the mode of action appears to be as fellows: under diabetic condition, ROS production is increased, JNK is activated, and Akt activity is inhibited; TO-mediated LXR activation potently inhibits ROS production, increases anti-oxidative gene expressions, suppresses JNK activation, and restores Akt activity. Our data provide new evidence to support LXRs as promising therapeutic targets for anti-diabetic drug development.

The Pregnane X Receptor Attenuates Cytokine‐Induced Intestinal Epithelial Barrier Dysfunction, an Effect Associated With Inhibition of JNK Activation and Enhanced Autophagy

The Pregnane X Receptor Attenuates Cytokine‐Induced Intestinal Epithelial Barrier Dysfunction, an Effect Associated With Inhibition of JNK Activation and Enhanced Autophagy

Inhibition of c-Jun N-terminal kinase activation reverses Alzheimer disease phenotypes in APPswe/PS1dE9 mice.

Growing evidence indicates that the activation of c-Jun N-terminal kinase (JNK) is implicated in the multiple major pathological features of Alzheimer disease (AD). However, whether specific inhibition of JNK activation could prevent disease progression in adult transgenic AD models at moderate stage remains unknown. Here we first investigated the potential disease-modifying therapeutic effect of systemic administration of SP600125, a small-molecule JNK-specific inhibitor, in middle-aged APPswe/PS1dE9 mice. Using behavioral, histological, and biochemical methods, outcomes of SP600125 treatment on neuropathology and cognitive deficits were studied in APPswe/PS1dE9 mice. Compared with vehicle-treated APPswe/PS1dE9 mice, chronic treatment of SP600125 for 12 weeks potently inhibited JNK activation, which resulted in a marked improvement of behavioral measures of cognitive deficits and a dramatic reduction in amyloid plaque burden, β-amyloid production, tau hyperphosphorylation, inflammatory responses, and synaptic loss in these transgenic animals. In particular, we found that SP600125 treatment strongly promoted nonamyloidogenic amyloid precursor protein (APP) processing and inhibited amyloidogenic APP processing via regulating APP-cleavage secretase expression (ie, ADAM10, BACE1, and PS1) in APPswe/PS1dE9 mice. Our findings demonstrate that chronic SP600125 treatment is powerfully effective in slowing down disease progression by markedly reducing multiple pathological features and ameliorating cognitive deficits associated with AD. This study highlights the concept that active JNK actually contributes to the development of the disease, and provides critical preclinical evidence that specific inhibition of JNK activation by SP600125 treatment may be a novel promising disease-modifying therapeutic strategy for the treatment of AD.

Metformin protects primary rat hepatocytes against oxidative stress-induced apoptosis.

The majority of chronic liver diseases are accompanied by oxidative stress, which induces apoptosis in hepatocytes and liver injury. Recent studies suggest that oxidative stress and insulin resistance are important in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and the pathophysiology of diabetes complications. Metformin has been shown to be hepatoprotective in the insulin-resistant and leptin-deficient ob/ob mouse model of NAFLD. However, the mechanism involved in the protective effects of metformin has not been elucidated yet. Therefore, we investigated the protective effect of metformin against oxidative stress-induced apoptosis. Primary rat hepatocytes were exposed to the oxidative stress-generating compound menadione in the presence and absence of metformin. Apoptosis was determined by measuring caspase activity and poly(ADP-ribose) polymerase (PARP)-cleavage, and necrosis was measured by Sytox Green nuclear staining. We demonstrate that (1) Metformin inhibits menadione-induced caspase-9,-6,-3 activation and PARP-cleavage in a concentration-dependent manner. (2) Metformin increases menadione-induced heme oxygenase-1 (HO-1) expression and inhibits c-Jun N-terminal kinase (JNK)-phosphorylation. (3) Metformin does not induce necrosis in primary hepatocytes. Metformin protects hepatocytes against oxidative stress-induced caspase activation, PARP-cleavage and apoptosis. The anti-apoptotic effect of metformin is in part dependent on HO-1 and bcl-xl induction and inhibition of JNK activation and independent of insulin signaling. Our results elucidate novel protective mechanisms of metformin and indicate that metformin could be investigated as a novel therapeutic agent for the treatment of oxidative stress-related liver diseases.

Short time tripterine treatment enhances endothelial progenitor cell function via heat shock protein 32.

The dysfunction of endothelial progenitor cells (EPCs) limits their potential for the treatment of ischemia and atherosclerosis. Therefore, we investigated the effect of tripterine on EPC function and examined the underlying mechanisms. The effect of tripterine, an active component of Tripterygium wilfordii Hook, on the enhancement of EPC function and the efficiency of EPC transplantation was investigated in vitro and in vivo. Treatment of EPCs with tripterine at 2.5 µM for 4 h inhibited oxidized low-density lipoprotein (ox-LDL) induced ROS production, cell apoptosis, and cell senescence and improved the migration and tube formation capacities of EPCs treated with ox-LDL (200 µg/ml). In vivo studies showed that tripterine conditioning of EPCs administered to ischemic foci improved blood perfusion and microvascular density in a mouse hindlimb ischemia model. Examination of the underlying mechanisms indicated that the effect of tripterine is mediated by the induction of heat shock protein 32 expression and the inhibition of JNK activation. The present results are of clinical significance because they suggest the potential of tripterine as a therapeutic agent to improve the efficacy of EPC transplantation for the treatment of ischemic diseases.

Oestrogen inhibits BMP4‐induced BMP4 expression in cardiomyocytes: a potential mechanism of oestrogen‐mediated protection against cardiac hypertrophy

Oestrogen inhibits cardiac hypertrophy and bone morphogenetic protein-4 (BMP4) induces cardiac hypertrophy. Here we have studied the inhibition by oestrogen of BMP4 expression in cardiomyocytes. Cultures of neonatal rat cardiomyocytes were used in in vitro experiments. Bilatαl ovariectomy (OVX) was carried out in female Kunming mice and cardiac hypertrophy was induced by transverse aortic constriction (TAC). Oestrogen inhibited BMP4-induced cardiomyocyte hypertrophy and BMP4 expression in vitro. The inhibition of BMP4-induced BMP4 protein expression by oestrogen was prevented by the inhibitor of oestrogen receptor-β, PHTPP, but not by the inhibitor of oestrogen receptor-α MPP. BMP4 induced smad1/5/8 activation, which was not affected by oestrogen in cardiomyocytes. BMP4 induced JNK but not ERK1/2 and p38 activation, and activated JNK was inhibited by oestrogen. Treatment with the p38 inhibitor SB203580 or the JNK inhibitor SP600125 inhibited BMP4-induced BMP4 expression in cardiomyocytes, but the ERK1/2 inhibitor U0126 increased BMP4-induced BMP4 expression, indicating that JNK, ERK1/2 and p38 MAPKs were all involved, although only JNK activation contributed to the inhibition of BMP4-induced BMP4 expression by oestrogen. TAC induced significant heart hypertrophy in OVX mice in vivo and oestrogen replacement inhibited TAC-induced heart hypertrophy in OVX mice. In parallel with the data of heart hypertrophy, oestrogen replacement significantly reduced the increased BMP4 protein expression in TAC-treated OVX mice. Oestrogen treatment inhibited BMP4-induced BMP4 expression in cardiomyocytes through stimulating oestrogen receptor-β and inhibiting JNK activation. Our results provide a novel mechanism underlying oestrogen-mediated protection against cardiac hypertrophy.

Activity of ABT-199 and Acquired Resistance in Follicular Lymphoma Cells

ABT-199, a platelet-sparing pro-apoptotic BH3 mimetic shows promise in the treatment of B-cell malignancy such as chronic lymphocytic leukemia (CLL). Given the central role of BCL2 expression in follicular lymphoma (FL), we studied the effects of ABT-199 on proliferation, induction of apoptosis, and signaling pathways in the t(14;18)+ FL cell lines WSU-FSCCL and FC-TxFL2 and in primary FL cells. We also developed an ABT-199-resistant FC-TxFL2 cell line by continuous treatment with ABT-199.The growth of tested cell lines and primary FL cells was significantly inhibited with nanomolar concentrations of ABT-199. FC-TxFL2 cells (IC50 = 7 nM) were more sensitive to ABT-199 treatment than WSU-FSCCL cells (IC50 = 110 nM). The increased sensitivity was associated with higher levels of BIM pro-apoptotic protein. ABT-199 induced rapid phosphorylation of JNK kinase and substantial decrease of mitochondrial potential in FC-TxFL2 cells. Inhibition of JNK activation further augmented the apoptosis induced with ABT-199. All other tested anti-apoptotic (such as BCL-2, BCL-xL, MCL-1) and pro-apoptotic proteins (such as Bax, Puma, Bak, Bad, Noxa, Bid, Bok) were similarly expressed in both cell lines and did not change upon ABT-199 treatment. Surprisingly, cleavage of Bid was observed, which points also to activation of extrinsic apoptotic pathway. In cells with acquired resistance after continuous ABT-199 exposure, we identified increased levels of MCL-1, and elevated phosphorylation of BCL-2 on T56 and of AKT on S473, but also evidence for increased autophagy, as assessed by LC3BI/II expression, compared to parental cells. These cells remained resistant several weeks after omitting ABT-199 from culture medium.In a FL murine xenograft model, ABT-199 showed anti-tumor activity. Treatment significantly decreased tumors volumes compared to untreated mice. Subsequently isolated xenograft FC-TxFL2 cells previously exposed to ABT-199 showed increased resistance to ABT-199. However, unlike in vitro exposed cells, the resistance quickly diminished after cultivation without ABT-199. Next, the ability to overcome ABT-199 resistance with epigenetic modifiers such as decitabine and vorinostat was tested. A strong synergistic effect in potentiation of apoptosis with these therapeutics was observed.Despite the theoretic rationale for BH3 mimetic activity in FL, activity in patients with FL has not been as dramatic as has been observed for CLL. Relatively easily acquired resistance observed in our model may explain this failure. Therefore, combinations that can prevent resistance acquisition should be intensively studied and moved quickly to clinical trials. DisclosuresSmith:Abbvie: Research Funding.

Abstract 13780: JNK Inhibition Mimics the Beneficial Effect of Roux-en-Y Gastric Bypass Surgery on Obesity-Induced Endothelial Dysfunction

Introduction: Roux-en-Y gastric bypass surgery (RYGB) reduces weight and long-term cardiovascular risk in obese patients. We previously demonstrated that in diet-induced obese rats RYGB improves aortic endothelial vasorelaxation 8 days after surgery in a weight loss-independent manner. Improved endothelial function was associated with decreased JNK phosphorylation and increased Akt and eNOS phosphorylation in the aorta. Hypothesis: We investigated whether in vivo inhibition of JNK activity in sham-operated ad libitum-fed rats mimics these endothelial effects of RYGB. Methods: Male Wistar rats were fed a high fat (60%) high cholesterol (1.25%) diet for 7 weeks before undergoing either RYGB or sham surgery; sham-operated rats received vehicle (sham_AL) or the JNK-specific inhibitor SP600125 40mg/kg/day s.c. for 8 days post-surgery (sham_SP). Then, thoracic aortic rings were isolated and subjected to ex vivo isometric tension recordings. After submaximal contraction with norepinephrine (10-6mol/L), cumulative relaxation responses were performed to GLP-1 (7-36) amide (10-12 to 10-6mol/L) or insulin (10-12 to 10-5mol/L); tests were repeated after preincubation with the eNOS inhibitor L-NAME (10-4mol/L). Western blot analysis of JNK, Akt and eNOS was also performed on aortic tissue lysates. Results: Body weight did not differ between sham_SP and sham_AL rats, while the weight loss of RYGB rats was significant 8 days after surgery. GLP-1- and insulin-induced vasorelaxation responses improved in RYGB compared to sham_AL rats; interestingly, sham_SP rats had endothelial relaxation similar to RYGB rats. All responses were blocked by preincubation with L-NAME, suggesting an eNOS-dependent effect. JNK protein phosphorylation in aortic lysates was decreased in RYGB and sham_SP rats compared to sham_AL, while Akt phosphorylation was increased. eNOS phosphorylation and dimerization were also increased in RYGB and sham_SP compared to sham_AL rats. Conclusion: JNK inhibition seems to mimic the immediate eNOS-mediated endothelial protective effects of RYGB. Further, our study underlines a crucial role of JNK in obesity-associated endothelial dysfunction, suggesting a novel mechanism for the cardiovascular effects of RYGB.

Metformin impairs Rho GTPase signaling to induce apoptosis in neuroblastoma cells and inhibits growth of tumors in the xenograft mouse model of neuroblastoma.

Metformin has been shown to inhibit tumor growth in xenograft rodent models of adult cancers, and various human clinical trials are in progress. However, the precise molecular mechanisms of metformin action are largely unknown. In the present study we examined the anti-tumor activity of metformin against neuroblastoma, and determined the underlying signaling mechanisms. Using human neuroblastoma xenograft mice, we demonstrated that oral administration of metformin (100 and 250 mg/kg body weight) significantly inhibited the growth of tumors. The interference of metformin in spheroid formation further confirmed the anti-tumor activity of metformin. In tumors, the activation of Rac1 (GTP-Rac1) and Cdc42 (GTP-Cdc42) was increased while RhoA activation (GTP-RhoA) was decreased by metformin. It also induced phosphorylation of JNK and inhibited the phosphorylation of ERK1/2 without affecting p38 MAP Kinase. Infection of cells by adenoviruses expressing dominant negative Rac1 (Rac1-N17), Cdc42 (Cdc42-N17) or constitutively active RhoA (RhoA-V14), or incubation of cells with pharmacological inhibitors of Rac1 (NSC23766) or Cdc42 (ML141) significantly protected neuroblastoma cells from metformin-induced apoptosis. Additionally, inhibition of JNK activity along with Rac1 or Cdc42 attenuated cytotoxic effects of metformin. These studies demonstrated that metformin impairs Rho GTPases signaling to induce apoptosis via JNK pathway.