Activation Of c-Jun Research Articles

Abstract 2855: Oleic acid-induced ANGPTL4 facilitates metastasis of human colorectal cancer via up-regulation of NOX4 expression

Abstract Colorectal cancer (CRC) is highly associated with metabolic diseases, such as obesity and diabetes. Elevated free fatty acids (FFAs) in cancer patients with metabolic disorders may be associated with cancer progression. In addition, increased levels of reactive oxygen species (ROS) are also observed in dyslipidemia which induces expression of NADPH oxidase 4 (NOX4) by FFAs in aorta, kidney, and white adipocyte in physiology condition. Although the expression of NOX4 has been also up-regulated in various cancer cell types, the correlation between up-regulation of NOX4 and dyslipidemia-regulated CRC metastasis remains unclear. Here, we found that oleic acid (OA) induced-angiopoietin-like 4 (ANGPTL4) expression was through the activation of PPARs pathways, resulting in promoting the metastasis of CRC. The recombinant protein of human ANGPTL4 rescued the OA-induced invasive ability in ANGPTL4 knockdown cells. It is worthy to note that the knockdown of ANGPTL4 not only significantly inhibited OA-induced NOX4 expression, but also reduced ROS production. In addition, the depletion of ROS by using NAC or knockdown of NOX4 inhibited OA-induced extravasation of CRC cells in vivo. OA-induced MMP1 and MMP9 expressions were also dependent on the expression of ANGPTL4 and NOX4 in CRC cells. The transcriptional activation of NOX4 gene by OA-induced ANGPTL4 was regulated by activation of c-Jun and dependent on AP-1 site of NOX4 promoter. These results reveal that OA-promoted CRC metastasis was through the activation of ANGPTL4/NOX4 axis, suggesting that ANGPTL4 and NOX4 may be potential therapeutic targets and diagnostic markers to improving outcomes for patients with CRC. Citation Format: Chih-Jie Shen, Yu-Han Liao, Jhih-Peng Tsai, Liang-Yi Hung, Wen-Chang Chang, Ben-Kuen Chen. Oleic acid-induced ANGPTL4 facilitates metastasis of human colorectal cancer via up-regulation of NOX4 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2855.

Abstract 3800: HDAC8 regulates plasticity and escape from therapy in BRAF mutant melanoma

Abstract Melanoma cells are highly plastic and have the ability to switch to a dedifferentiated, invasive phenotype in response to multiple stimuli. We here show that exposure to melanoma cell lines and patient specimens to multiple stresses including BRAF-MEK inhibitor therapy, hypoxia and UV-irradiation leads to an increase in HDAC8 expression/activity, and in turn, the adoption of a drug-resistant, invasive phenotype. Systems level analyses using mass spectrometry-based phosphoproteomics and RNA-Seq demonstrated HDAC8 to be involved in pathways that regulated cell cycle entry, ribosome function, RNA binding, regulation of the cytoskeleton and MAPK pathway signaling. Introduction of HDAC8 into drug-naïve melanoma cells conveyed resistance in vitro and in in vivo xenograft models. The HDAC8-mediated BRAF inhibitor resistance was mediated through upstream receptor tyrosine kinase (RTK) activation, Ras/CRAF/MEK/ERK signaling and the suppression of apoptosis through increased stabilization of Mcl-1 and the inhibition of pro-apoptotic BIM. Among the multiple RTKs increased by HDAC8, EGFR emerged as a direct transcriptional target, and it was found that EGFR inhibition could overcome HDAC8-mediated tolerance to BRAF inhibition. Although it is known that HDACs primarily function at the histone level, they can also regulate signaling through the modulation of cytoplasmic protein acteylation. In line with this, we observed that HDAC8 introduction decreased the acetylation of c-JUN, leading to an increase in its transcriptional activity and the enrichment for an AP-1 gene signature. Mutation of putative acetylation sites in c-JUN (K268R, K271R, K273R) reduced the transcriptional activation of c-JUN in melanoma cells and conveyed resistance to BRAF inhibition through increased MAPK pathway activity. In vivo xenograft studies confirmed the key role of HDAC8 in therapeutic adaptation, with either a pan-HDAC inhibitor (panobinostat) or an HDAC8 inhibitor (PCI-34051) enhancing the durability of response to BRAF inhibitor therapy. We have thus identified HDAC8 as a key driver of phenotype switching in melanoma that regulates the responses to multiple cellular stresses and conveys resistance to BRAF inhibition. Our studies demonstrate that isoform-specific HDAC8 inhibitors could be an excellent strategy to limit the adaptation of melanoma cells to multiple stresses and therapeutic interventions, including the BRAF-MEK inhibitor combination. Citation Format: Michael Emmons, Fernanda Flores, John Koomen, Edward Seto, Jane Messina, Eric Lau, Jonathan Licht, Keiran Smalley. HDAC8 regulates plasticity and escape from therapy in BRAF mutant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3800.

Carbon Monoxide Releasing Molecule-2-Upregulated ROS-Dependent Heme Oxygenase-1 Axis Suppresses Lipopolysaccharide-Induced Airway Inflammation.

The up-regulation of heme oxygenase-1 (HO-1) is mediated through nicotinamaide adenine dinucleotide phosphate (NADPH) oxidases (Nox) and reactive oxygen species (ROS) generation, which could provide cytoprotection against inflammation. However, the molecular mechanisms of carbon monoxide-releasing molecule (CORM)-2-induced HO-1 expression in human tracheal smooth muscle cells (HTSMCs) remain unknown. Here, we found that pretreatment with CORM-2 attenuated the lipopolysaccharide (LPS)-induced intercellular adhesion molecule (ICAM-1) expression and leukocyte count through the up-regulation of HO-1 in mice, which was revealed by immunohistochemistrical staining, Western blot, real-time PCR, and cell count. The inhibitory effects of HO-1 by CORM-2 were reversed by transfection with HO-1 siRNA. Next, Western blot, real-time PCR, and promoter activity assay were performed to examine the HO-1 induction in HTSMCs. We found that CORM-2 induced HO-1 expression via the activation of protein kinase C (PKC)α and proline-rich tyrosine kinase (Pyk2), which was mediated through Nox-derived ROS generation using pharmacological inhibitors or small interfering ribonucleic acids (siRNAs). CORM-2-induced HO-1 expression was mediated through Nox-(1, 2, 4) or p47phox, which was confirmed by transfection with their own siRNAs. The Nox-derived ROS signals promoted the activities of extracellular signal-regulated kinase 1/2 (ERK1/2). Subsequently, c-Fos and c-Jun—activator protein-1 (AP-1) subunits—were up-regulated by activated ERK1/2, which turned on transcription of the HO-1 gene by regulating the HO-1 promoter. These results suggested that in HTSMCs, CORM-2 activates PKCα/Pyk2-dependent Nox/ROS/ERK1/2/AP-1, leading to HO-1 up-regulation, which suppresses the lipopolysaccharide (LPS)-induced airway inflammation.

JNK inhibition blocks piperlongumine-induced cell death and transcriptional activation of heme oxygenase-1 in pancreatic cancer cells.

Piperlongumine (PL) is an alkaloid that inhibits glutathione S-transferase pi 1 (GSTP1) activity, resulting in elevated reactive oxygen species (ROS) levels and cancer-selective cell death. We aimed to identify stress-associated molecular responses to PL treatment in pancreatic ductal adenocarcinoma (PDAC) cells. GSTP1 directly interacts with JNK, which is activated by oxidative stress and can lead to decreased cancer cell proliferation and cell death. Therefore, we hypothesized that JNK pathways are activated in response to PL treatment. Our results show PL causes dissociation of GSTP1 from JNK; robust JNK, c-Jun, and early ERK activation followed by suppression; increased expression of cleaved caspase-3 and cleaved PARP; and nuclear translocation of Nrf2 and c-Myc in PDAC cells. Gene expression analysis revealed PL caused a > 20-fold induction of heme oxygenase-1 (HO-1), which we hypothesized was a survival mechanism for PDAC cells under enhanced oxidative stress. HO-1 knockout resulted in enhanced PL-induced PDAC cell death under hypoxic conditions. Similarly, high concentrations of the HO-1 inhibitor, ZnPP (10µM), sensitized PDAC cells to PL; however, lower concentrations ZnPP (10nM) and high or low concentrations of SnPP both protected PDAC cells from PL-induced cell death. Interestingly, the JNK inhibitor significantly blocked PL-induced PDAC cell death, Nrf-2 nuclear translocation, and HMOX-1 mRNA expression. Collectively, the results demonstrate JNK signaling contributes to PL-induced PDAC cell death, and at the same time, activates Nrf-2 transcription of HMOX-1 as a compensatory survival mechanism. These results suggest that elevating oxidative stress (using PL) while at the same time impairing antioxidant capacity (inhibiting HO-1) may be an effective therapeutic approach for PDAC.

HDAC8 Regulates a Stress Response Pathway in Melanoma to Mediate Escape from BRAF Inhibitor Therapy.

Melanoma cells have the ability to switch to a dedifferentiated, invasive phenotype in response to multiple stimuli. Here, we show that exposure of melanomas to multiple stresses including BRAF-MEK inhibitor therapy, hypoxia, and UV irradiation leads to an increase in histone deacetylase 8 (HDAC8) activity and the adoption of a drug-resistant phenotype. Mass spectrometry-based phosphoproteomics implicated HDAC8 in the regulation of MAPK and AP-1 signaling. Introduction of HDAC8 into drug-naïve melanoma cells conveyed resistance both in vitro and in vivo. HDAC8-mediated BRAF inhibitor resistance was mediated via receptor tyrosine kinase activation, leading to MAPK signaling. Although HDACs function at the histone level, they also regulate nonhistone substrates, and introduction of HDAC8 decreased the acetylation of c-Jun, increasing its transcriptional activity and enriching for an AP-1 gene signature. Mutation of the putative c-Jun acetylation site at lysine 273 increased transcriptional activation of c-Jun in melanoma cells and conveyed resistance to BRAF inhibition. In vivo xenograft studies confirmed the key role of HDAC8 in therapeutic adaptation, with both nonselective and HDAC8-specific inhibitors enhancing the durability of BRAF inhibitor therapy. Our studies demonstrate that HDAC8-specific inhibitors limit the adaptation of melanoma cells to multiple stresses including BRAF-MEK inhibition. SIGNIFICANCE: This study provides evidence that HDAC8 drives transcriptional plasticity in melanoma cells in response to a range of stresses through direct deacetylation of c-Jun.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2947/F1.large.jpg.

N-acetyl-cysteine blunts 6-hydroxydopamine- and L-buthionine-sulfoximine-induced apoptosis in human mesenchymal stromal cells.

Parkinson disease (PD) is characterized by the loss of dopaminergic (DAergic) neurons linked to environmental toxicants that cause oxidative stress (OS). The aim of this investigation was to establish the molecular response of human mesenchymal stroma cells (MSCs) depleted of glutathione (GSH) by the specific inhibitor L-buthionine-sulfoximine (BSO) to 6-hydroxydopamine (6-OHDA) and/or N-acetylcysteine (NAC) co-treatment. We found that treatment with BSO (10mM) plus 6-OHDA (200μM) induced apoptosis in MSCs through an oxidative stress (OS) mechanism involving H2O2, reflected by the detection of dichlorofluorescein-positive (DCF+) cells and oxidation of DJ-1 Cys106-SH into DJ-1 Cys106-SO3; an almost complete reduction in glutathione peroxidase 1 (GPX1) expression; activation of the transcription factor c-JUN, the pro-apoptotic protein BAX and BH-3-only protein PUMA; loss of mitochondrial membrane potential (∆Ψm); activation of the protease caspase-3 (CASP3) and apoptosis-inducing factor (AIF); chromatin condensation; and DNA fragmentation. Strikingly, co-treatment of MSCs with NAC (5mM) and BSO + 6-OHDA significantly reduced the expression of OS and cell death markers but were unable to restore the expression of GPX1 compared to the expression in untreated or treated cells with NAC only. These findings highlighted the importance of the maintenance of the GSH-dependent (e.g., GPX1, GSH synthesis) and -independent (e.g., ROS scavenger molecules and thiol reducing activity) antioxidant systems (e.g., NAC) in the protection of MSCs from detrimental stress stimuli, thereby increasing the survival of stromal cells.

Oncofetal HLF transactivates c-Jun to promote hepatocellular carcinoma development and sorafenib resistance

Background and aimsThe unique expression pattern makes oncofetal proteins ideal diagnostic biomarkers and therapeutic targets in cancer. However, few oncofetal proteins have been identified and entered clinical practice.MethodsFetal liver, adult...

Chondroprotective effects of platelet lysate towards monoiodoacetate-induced arthritis by suppression of TNF-α-induced activation of NF-ĸB pathway in chondrocytes.

Platelet lysate (PL) contains a cocktail of growth factors that actively participates in cartilage repair. This study was designed to determine the effect and mechanism of PL on osteoarthritis (OA). An arthritis model was established to mimic human OA by intra-articular injection of monoiodoacetate (MIA) to Sprague Dawley (SD) rats. The model was weekly treated with PL by intra-articular injection. Thermal withdrawal latency, mechanical withdrawal threshold, and treadmill gait were tested for pain behavior observation. Histopathological and immunohistochemical analyses were conducted for evaluating cartilage degradation. Real time PCRs and Western blots were conducted to elucidate the mechanism of PL on primary chondrocytes. Results showed that, in vivo, PL significantly attenuated pain symptoms and exerted chondrocyte-protective and extracellular matrix (ECM)-modifying effect on the arthritic cartilage in a dose-dependent manner. The in situ expressions of type II Collagen (Col2) and matrix metalloproteinase 13 (Mmp13) in the arthritic cartilage was abnormal and was restored by PL. In vitro, PL significantly restored tumor necrosis factor α (TNF-α)-suppressed anabolic gene expression (Col2 and aggrecan) and TNF-α-increased catabolic gene expression (Col10, Mmp13, Adamts5, and Adamts9) in chondrocytes. The effects were mediated by TNF-α downstream signaling, including inhibition of NF-κB and c-Jun activities. This study provides certain knowledge of anti-OA effect and TNF signaling-related mechanism of PL, placing it as a promising and alternative option for OA therapy in the future.

An in silico study on plant-derived inhibitors against a prognostic Biomarker, Jab1

Cancer kills millions of people worldwide every year. The main form of treatment at this point is chemotherapy, which comprises of systemic drug delivery so that they can kill the cancerous cells more effectively. But most of these drugs cause severe side effects in patients and, therefore there is a strong need to focus on identifying natural compounds as a potent phytoinhibitors using various in silico and in vitro approaches. Natural compounds pose low toxicity, hence render them to be an excellent alternative to the basis for the development of new anti-cancerous drugs. Our study considers an effective therapeutic target, Jab1 (c-Jun activation domain-binding protein-1) or a c-Jun coactivator, which has been implicated in multiple protein interactions that play a significant role in various stages of carcinogenesis. Hence we have performed screening of 1500 natural compounds having anticancerous activity by applying various in silico approaches including Lipinski rule of five, ADME, and various Molecular Docking tools. In this study, we have identified two potent phytoinhibitors against Jab1 which needs to be further validated through in vitro approaches.

Endothelin‐1 Induced Fibronectin Expression via c‐Jun

Glaucoma is an optic disease that is characterized by slow neurodegeneration of the optical nerves and steady loss of retinal ganglion cells. Over 70 million people suffer from glaucoma and it is estimated that over 118 million will suffer from it in 2040. A prime factor causing glaucoma is an increased in intraocular pressure (IOP) but the mechanism in how IOP contributes to glaucoma is still being studied. Blocking the aqueous humor through the trabecular meshwork (TM) is a major contributor for the IOP elevation. Endothelin (ET‐1) is a potent vasoconstrictor in which play's an important role in vascular system. Previous research has discovered that ET‐1 and its ETA/ETB receptors are also involved in the pathogenesis of glaucoma. The current study aims to test whether ET‐1 induced fibronectin deposition at TM. We hypothesize that ET‐1 induces over expression of fibronectin through c‐Jun‐mediated signaling pathway. Primary human TM cells were pretreated with c‐Jun siRNA, SP600125 (a JNK inhibitor to block activation of c‐Jun) or ET receptor antagonist's followed with ET‐1 treatment. Protein levels of fibronectin and c‐Jun were detected using immunoblotting. An increase of fibronectin and c‐Jun were detected in TM cells treated with ET‐1 for 24–72 hours. Fibronectin was increased as a result of c‐Jun being partially terminated by the knockdown of c‐Jun by C‐Jun siRNA. Blocking of ETA/ETB by BQ610/BQ788 reduced the expression of fibronectin. Our results preliminary demonstrate that ET‐1 induces the expression of fibronectin through c‐Jun mediated signaling pathway, suggesting that ET‐1 may cause the accumulation of fibronectin at TM contributing to IOP elevation.Support or Funding InformationThis work was supported by the Department of Health and Human Services, National Institute of Health, National Heart, Lung and Blood Institute, SMART Award Number 4R25HL007786‐25 to Harlan Jones, Ph.D This work was also supported by MARC U*STARGrant5T34GM008395 to MariaElena Zavala, Ph.DThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A nonsense mutant of the hepatitis B virus large S protein antagonizes multiple tumor suppressor pathways through c-Jun activation domain-binding protein1.

Hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC). Previous studies have identified recurrent nonsense mutations in the HBV large S (LHBs) gene from the liver from HBV core antigen-positive HCC patients. These nonsense mutants have been shown to be oncogenic in mouse xenograft models using a mouse embryonic fibroblast cell line. Here, we expressed in a liver cell line Huh-7 a carboxy terminally truncated protein from a nonsense mutant of the LHBs gene, sW182* (stop codon at tryptophane-182). Although the sW182* protein appeared not to be very stable in the cultured liver cells, we confirmed that the protein can be highly expressed and retained for a prolonged period of time in the hepatocytes in the mouse liver, indicating its stable nature in the physiological condition. In the Huh-7 cells, the sW182* mutant downregulated tumor suppressors p53 and Smad4. This downregulation was reversed by a proteasome inhibitor MG132, implying the involvement of proteasome-based protein degradation in the observed regulation of the tumor suppressors. On the other hand, we found that c-Jun activation domain-binding protein 1 (Jab1) physically interacts with the sW182*, but not wild-type LHBs. RNA interference (RNAi) of Jab1 restored the levels of the downregulated p53 and Smad4. The sW182* mutant inhibited the promoter activity of downstream target genes of the tumor suppressors. Consistently, Jab1 RNAi reversed the inhibition. These results suggest that the LHBs nonsense mutant antagonizes the tumor suppressor pathways through Jab1 in the liver contributing to HCC development.

Fine particulate matter (PM2.5) inhibits ciliogenesis by increasing SPRR3 expression via c-Jun activation in RPE cells and skin keratinocytes

Exposure to fine particulate matter (PM) with diameter <2.5 µm (PM2.5) causes epithelium injury and endothelial dysfunction. Primary cilia are sensory organelles that transmit extracellular signals into intracellular biochemical responses and have roles in physiology. To date, there have been no studies investigating whether PM2.5 affects primary cilia in skin. We addressed this in the present study using normal human epidermal keratinocytes (NHEKs) and retinal pigment epithelium (RPE) cells. We found that formation of primary cilium is increased in differentiated NHEKs. However, treatment with PM2.5 blocked increased ciliogenesis in NHEKs and RPE cells. Furthermore, PM2.5 transcriptionally upregulated small proline rich protein 3 (SPRR3) expression by activating c-Jun, and ectopic expression of SPRR3 inhibits suppressed the ciliogenesis. Accordingly, treatment with c-Jun activator (anisomycin) induced SPRR3 expression, whereas the inhibitor (SP600125) recovered the ciliated cells and cilium length in PM2.5-treated cells. Moreover, c-Jun inhibitor suppressed upregulation of SPRR3 in PM2.5-treated cells. Taken together, our finding suggested that PM2.5 inhibits ciliogenesis by increasing SPRR3 expression via c-Jun activation in RPE cells and keratinocytes.

MKP2 suppresses TGF-β1-induced epithelial-to-mesenchymal transition through JNK inhibition.

Interstitial fibrosis is a typical feature of end-stage renal diseases, regardless of the initial cause of kidney injury. Epithelial-to-mesenchymal transition (EMT) is a mechanism that is thought to play a role in generating the interstitial matrix-producing myofibroblasts and is prominently induced by the transforming growth factor-β 1 (TGF-β1). TGF-β1 signals through a variety of Smad and non-Smad signaling pathways, including the mitogen-activated protein kinase (MAPK) pathways. In a study published in a recent issue of Clinical Science (Clin. Sci. (2018) 132(21),2339-2355), Li et al. investigated the potential role of the Mitogen-activated protein kinase phosphatase 2 (MKP2), also known as Dusp4, in the control of EMT and renal fibrosis. Based on results obtained with an animal model of kidney fibrosis and a proximal tubular epithelial cell line system, the authors put forward a role for MKP2 as a negative feedback regulator of TGF-β1-induced EMT and fibrosis in the kidney. Intriguingly, MKP2 is found to down-regulate activity of c-Jun, but not that of other MAPKs, extracellular signal-regulated kinases or p38, implying a role for c-Jun N-terminal kinase-dependent signaling in renal fibrosis. In this commentary, I discuss the findings of Li and co-workers in the context of the recent literature placing a focus on potential clinical/therapeutic implications.

Rsu1-dependent control of PTEN expression is regulated via ATF2 and cJun.

The Rsu1 protein contributes to cell adhesion and migration via its association with the adaptor complex of Integrin linked kinase (ILK), PINCH, and Parvin (IPP), which binds to the cytoplasmic domain of β1 integrins joining integrins to the actin cytoskeleton. Rsu1 binding to PINCH in the IPP complex is required for EGF-induced adhesion, spreading and migration in MCF10A mammary epithelial cells. In addition, Rsu1 expression inhibits Jun kinase but is necessary for the activation of MKK4 and p38 Map kinase signaling essential for migration in MCF10A cells. The data reported here examines the links between MKK4-p38-ATF2 signaling and AKT regulation in MCF10A cells. Ectopic Rsu1 inhibited AKT1 phosphorylation while Rsu1 depletion induced AKT activation and AKT1 phosphorylation of MKK4 on serine 80, blocking MKK4 activity. Rsu1 depletion also reduced the RNA for lipid phosphatase PTEN thus implicating PTEN in modulating levels of activated AKT in these conditions. ChIP analysis of the PTEN promoter revealed that Rsu1 depletion prevented binding of ATF2 to a positive regulatory site in the PTEN promoter and the enhanced binding of cJun to a negatively regulatory PTEN promoter site. These results demonstrate a mechanism by which Rsu1 adhesion signaling alters the balance between MKK4-p38-ATF2 and cJun activation thus altering PTEN expression in MCF10A cells.

Acetaminophen hepatotoxicity: A mitochondrial perspective.

Acetaminophen (APAP) is a highly effective analgesic, which is safe at therapeutic doses. However, an overdose can cause hepatotoxicity and even liver failure. APAP toxicity is currently the most common cause of acute liver failure in the United States. Decades of research on mechanisms of liver injury have established the role of mitochondria as central players in APAP-induced hepatocyte necrosis and this chapter examines the various facets of the organelle's involvement in the process of injury as well as in resolution of damage. The injury process is initiated by formation of a reactive metabolite, which binds to sulfhydryl groups of cellular proteins including mitochondrial proteins. This inhibits the electron transport chain and leads to formation of reactive oxygen species, which induce the activation of redox-sensitive members of the MAP kinase family ultimately causing activation of c-Jun N terminal kinase, JNK. Translocation of JNK to the mitochondria then amplifies mitochondrial dysfunction, ultimately resulting in mitochondrial permeability transition and release of mitochondrial intermembrane proteins, which trigger nuclear DNA fragmentation. Together, these events result in hepatocyte necrosis, while adaptive mechanisms such as mitophagy remove damaged mitochondria and minimize the extent of the injury. This oscillation between recovery and necrosis is predominant in cells at the edge of the necrotic area in the liver, where induction of mitochondrial biogenesis is important for liver regeneration. All these aspects of mitochondria in APAP hepatotoxicity, as well as their relevance to humans with APAP overdose and development of therapeutic approaches will be examined in detail in this chapter.

Berberis lycium Royle fruit extract mitigates oxi-inflammatory stress by suppressing NF-κB/MAPK signalling cascade in activated macrophages and Treg proliferation in splenic lymphocytes.

Although Berberis plant species have been advocated as immune modulators, information regarding their mechanism(s) of action is limited. Therefore, in the present study we assessed the efficacy of Berberis lycium Royle fruit extract (BLFE) in the attenuation of lipopolysaccharide (LPS)-induced oxi-inflammatory aggravation and concanavalin A (Con-A)-induced proliferation in murine peritoneal macrophages and lymphocytes, respectively. BLFE strongly suppressed production of the oxidative and inflammatory effector molecules nitric oxide (NO), reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS), inflammatory cytokines (TNF-α/IL-6/IL-1β/IFN-γ) as well as chemokines (MCP-1 and RANTES), with a concomitant enhancement in heme oxygenase-1 (HO-1) and IL-10 levels. Subsequent mechanistic analysis revealed that BLFE strongly inhibited the phosphorylation of IκBα as well as MAPKs such as extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun NH2-terminal kinase (JNK), thereby directly resulting in the suppression of nuclear factor-κB (NF-ĸB) and c-Jun activation, ultimately culminating in the observed attenuation of inflammatory molecules. Additionally, BLFE appeared to mitigate Con-A-induced proliferation of Tregs (CD3+ CD4+ CD25+) thereby suggesting its modulatory effects on adaptive immune cells. UPLC-DAD-ESI-QTOF-MS/MS of BLFE revealed the presence of major bioactive phenolics and alkaloids including chlorogenic acid, rutin, catechin and quercetin 3-D-galactoside, berberine and magnoflorine, which could have synergistically contributed to the above findings. Overall, this work provides evidence that BLFE may be effective in the mitigation of inflammatory disorders, especially those associated with NF-κB/MAPK activation.

Oncolytic adenovirus Delta-24-RGD induces a widespread glioma proteotype remodeling during autophagy

Adenovirus Delta-24-RGD has shown a remarkable efficacy in a phase I clinical trial for glioblastoma. Delta-24-RGD induces autophagy in glioma cells, however, the molecular derangements associated with Delta-24-RGD infection remains poorly understood. Here, proteomics was applied to characterize the glioma metabolic disturbances soon after Delta-24-RGD internalization and late in infection. Minutes post-infection, a rapid survival reprogramming of glioma cells was evidenced by an early c-Jun activation and a time-dependent dephosphorylation of multiple survival kinases. At 48 h post-infection (hpi), a severe intracellular proteostasis impairment was characterized, detecting differentially expressed proteins related to mRNA splicing, cytoskeletal organization, oxidative response, and inflammation. Specific kinase-regulated protein interactomes for Delta-24-RGD-modulated proteome revealed interferences with the activation dynamics of protein kinases C and A (PKC, PKA), tyrosine-protein kinase Src (c-Src), glycogen synthase kinase-3 (GSK-3) as well as serine/threonine-protein phosphatases 1 and 2A (PP1, PP2A) at 48hpi, in parallel with adenoviral protein overproduction. Moreover, the late activation of the nuclear factor kappa B (NF-κB) correlates with the extracellular increment of specific cytokines involved in migration, and activation of different inflammatory cells. Taken together, our integrative analysis provides further insights into the effects triggered by Delta-24-RGD in the modulation of tumor suppression and immune response against glioma. SignificanceThe current study provides new insights regarding the molecular mechanisms governing the glioma metabolism during Delta-24-RGD oncolytic adenoviral therapy. The compilation and analysis of intracellular and extracellular proteomics have led us to characterize: i) the cell survival reprogramming during Delta-24-RGD internalization, ii) the proteostatic disarrangement induced by Delta-24-RGD during the autophagic stage, iii) the protein interactomes for Delta-24-RGD-modulated proteome, iv) the regulatory effects on kinase dynamics induced by Delta-24-RGD late in infection, and v) the overproduction of multitasking cytokines upon Delta-24-RGD treatment. We consider that the quantitative molecular maps generated in this study may establish the foundations for the development of complementary adenoviral based-vectors to increase the potency against glioma.

Requirement of Rab21 in LPS-induced TLR4 signaling and pro-inflammatory responses in macrophages and monocytes

Lipopolysaccharide (LPS) induces macrophage/monocyte activation and pro-inflammatory cytokines production by activating Toll-like receptor 4 (TLR-4) signaling. Rab GTPase 21 (Rab21) is a member of the Rab GTPase subfamily. In the present study, we show that LPS induced TLR4 and Rab21 association and endosomal translocation in murine bone marrow–derived macrophages (BMDMs) and primary human peripheral blood mononuclear cells (PBMCs). In BMDMs, shRNA-mediated stable knockdown of Rab21 inhibited LPS-induced expression and production of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α). Conversely, forced overexpression of Rab21 by an adenovirus construct potentiated LPS-induced IL-1β, IL-6 and TNF-α production in BMDMs. Further studies show that LPS-induced TLR4 endosomal traffic and downstream c-Jun and NFκB (nuclear factor-kappa B) activation were significantly inhibited by Rab21 shRNA, but intensified with Rab21 overexpression in BMDMs. Finally, in the primary human PBMCs, siRNA-induced knockdown of Rab21 significantly inhibited LPS-induced IL-1β, IL-6 and TNF-α production. Taken together, we suggest that Rab21 regulates LPS-induced pro-inflammatory responses by promoting TLR4 endosomal traffic and downstream signaling activation.

The c-Jun and JunB transcription factors facilitate the transit of classical Hodgkin lymphoma tumour cells through G1

Classical Hodgkin Lymphoma (cHL) is primarily a B cell lymphoid neoplasm and a member of the CD30–positive lymphomas. cHL and the other CD30–positive lymphomas are characterized by the elevated expression and/or constitutive activation of the activator protein-1 (AP-1) family transcription factors, c-Jun and JunB; however, the specific roles they play in the pathobiology of cHL are unclear. In this report we show that reducing either c-Jun or JunB expression with short-hairpin RNAs (shRNAs) reduced the growth of cHL cell lines in vitro and in vivo, primarily through impairing cell cycle transition through G1. We further investigated the effect of c-Jun and JunB knock-down on proliferation in another CD30–positive lymphoma, anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL). We found that JunB knock-down in most ALK+ ALCL cell lines examined also resulted in reduced proliferation that was associated with a G0/G1 cell cycle defect. In contrast, c-Jun knock-down in multiple ALK+ ALCL cell lines had no effect on proliferation. In summary, this study directly establishes that both c-Jun and JunB play roles in promoting HRS cell proliferation. Furthermore, we demonstrate there are similarities and differences in c-Jun and JunB function between cHL and ALK+ ALCL.

Inhibition of skin tumor promotion by TPA using a combination of topically applied ursolic acid and curcumin.

Prevention remains an important strategy to reduce the burden of cancer. One approach to prevent cancer is the use of phytochemicals in various combinations as safe and effective cancer preventative agents. The purpose of this study was to examine the effects of the combination of ursolic acid (UA) and curcumin (Curc) for potential combinatorial inhibition of skin tumor promotion using the mouse two-stage skin carcinogenesis model. In short-term experiments, the combination of UA + Curc given topically prior to 12-O-tetradecanoylphorbol-13-acetate (TPA) significantly inhibited activation of epidermal EGFR, p70S6K, NF-κB p50, Src, c-Jun, Rb, and IκBα. Levels of c-Fos, c-Jun, and Cox-2 were also significantly reduced by the combination compared to the TPA treated group. The alterations in these signaling pathways by the combination of UA + Curc were associated with decreased epidermal proliferation as assessed by measuring BrdU incorporation. Significant effects were also seen with the combination on epidermal inflammatory gene expression and dermal inflammation, with the greatest effects on expression of IL-1β, IL-6, IL-22, and CXCL2. Furthermore, results from skin tumor experiments demonstrated that the combination of UA + Curc given topically significantly inhibited mouse skin tumor promotion by TPA to a greater extent than the individual compounds given alone. The greatest effects were seen on tumor free survival, tumor size, and tumor weight, although tumor incidence and multiplicity were also further reduced by the combination. These results demonstrate the potential cancer chemopreventive activity and mechanism(s) for the combination of UA + Curc.