Stress-associated Protein Kinase Research Articles

KMT2D deficiency enhances the anti-cancer activity of L48H37 in pancreatic ductal adenocarcinoma.

BACKGROUNDNovel therapeutic strategies are urgently needed for patients with a delayed diagnosis of pancreatic ductal adenocarcinoma (PDAC) in order to improve their chances of survival. Recent studies have shown potent anti-neoplastic effects of curcumin and its analogues. In addition, the role of histone methyltransferases on cancer therapeutics has also been elucidated. However, the relationship between these two factors in the treatment of pancreatic cancer remains unknown. Our working hypothesis was that L48H37, a novel curcumin analog, has better efficacy in pancreatic cancer cell growth inhibition in the absence of histone-lysine N-methyltransferase 2D (KMT2D).AIMTo determine the anti-cancer effects of L48H37 in PDAC, and the role of KMT2D on its therapeutic efficacy.METHODSThe viability and proliferation of primary (PANC-1 and MIA PaCa-2) and metastatic (SW1990 and ASPC-1) PDAC cell lines treated with L48H37 was determined by CCK8 and colony formation assay. Apoptosis, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) levels, and cell cycle profile were determined by staining the cells with Annexin-V/7-AAD, JC-1, DCFH-DA, and PI respectively, as well as flow cytometric acquisition. In vitro migration was assessed by the wound healing assay. The protein and mRNA levels of relevant factors were analyzed using Western blotting, immunofluorescence and real time-quantitative PCR. The in situ expression of KMT2D in both human PDAC and paired adjacent normal tissues was determined by immunohistochemistry. In vivo tumor xenografts were established by injecting nude mice with PDAC cells. Bioinformatics analyses were also conducted using gene expression databases and TCGA.RESULTSL48H37 inhibited the proliferation and induced apoptosis in SW1990 and ASPC-1 cells in a dose- and time-dependent manner, while also reducing MMP, increasing ROS levels, arresting cell cycle at the G2/M stages and activating the endoplasmic reticulum (ER) stress-associated protein kinase RNA-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/activating transcription factor 4 (ATF4)/CHOP signaling pathway. Knocking down ATF4 significantly upregulated KMT2D in PDAC cells, and also decreased L48H37-induced apoptosis. Furthermore, silencing KMT2D in L48H37-treated cells significantly augmented apoptosis and the ER stress pathway, indicating that KMT2D depletion is essential for the anti-neoplastic effects of L48H37. Administering L48H37 to mice bearing tumors derived from control or KMT2D-knockdown PDAC cells significantly decreased the tumor burden. We also identified several differentially expressed genes in PDAC cell lines expressing very low levels of KMT2D that were functionally categorized into the extrinsic apoptotic signaling pathway. The KMT2D high- and low-expressing PDAC patients from the TCGA database showed similar survival rates,but higher KMT2D expression was associated with poor tumor grade in clinical and pathological analyses.CONCLUSIONL48H37 exerts a potent anti-cancer effect in PDAC, which is augmented by KMT2D deficiency.

INFLUENCE OF CURCUMIN ON CYTOKINES CONTENT AND ANGIOTENSIN-CONVERTING ACTIVITY UNDER INTRAHIPPOCAMPUS ADMINISTRATION OF β-AMYLOID PEPTIDE IN RATS

Curcumin (diferuloylmethane) is a natural polyphenol which manifests compound effect on the body homeostasis; it negatively influences the NF-kB and АР-1 transcription factors, suppresses expression of cyclooxygenase-2, lipoxygenase, NO-synthase, matrix metalloproteinase-9, urokinase of plasminogen type activator, tumor necrosis factor (TNF), chemokines, molecules of cell adhesion and cycline D1; it inhibits expression of growth factor receptors and activity of stress-associated proteinkinase (JNK), protein tyrosine kinases, as well as other protein serine/threonine kinases [1–3]. Curcumin also acts as inhibitor of DNA-methyltransferase, therefore it is regarded as DNA hypomethylating agent. It establishes balance between activity of histone acetyltransferase and histone deacetyltransferase thus influencing the expression of certain genes. At last, curcumin modulates activity of micro-RNAs and their numerous target genes [4–5]. The above-mentioned effects of curcumin are accumulated in its anti-oxidation, anti-inflammation, anti-tumor and anti-amyloidogenic properties [6–11]. Considered recently as one of probable Alzheimer’s disease (AD) factors is aggregation of -amyloid peptide (А) to fibrils or deposits as the main pathogenetic event [12–15]. In particular, several studies showed that А is essentially accumulated in the brain areas (hippocampus and cortex), which conduce to obtaining and processing of information, and memory [16–18]. This peptide is formed during amyloidogenic processing of amyloid protein precursor (АРР) [19–21]. In a non-amyloidogenic way the full-size APP is decomposed by - and -secretases within Golgi apparatus and plasmatic membrane without forming the -amyloid peptide. Back internalization of a certain part of APP from the plasmatic membrane ant its transport towards the late endosomes results in - and -secretase-associated separation of А isoforms, 38 to 43 amino acid residues long [22]. The role of switch between non-amylodogenic and amyloidogenic ways of APP processing is

Abstract 547: TGFß Response Coupled to Stress Signaling by TGRL Lipolysis in Vascular Endothelial Cells

Elevation of circulating triglyceride-rich lipoproteins (TGRL) in the postprandial state is associated with increased endothelial cell inflammation and dysfunction potentially contributing to atherosclerosis. Previous studies from our laboratory showed that lipolysis products of TGRL induced endothelial cell barrier permeability, up-regulation of cytokines and adhesion molecules, and apoptosis. Gene array studies implicated the Stress Associated Protein Kinase and c-Jun N-terminal kinase (SAPK/JNK) pathway and identified the transcription factor activating transcription factor 3 (ATF3) as highly induced in this process. Additional studies demonstrated activation of second messengers for transforming growth factor beta (TGFβ) signaling (SMAD2/SMAD4) in response to lipolysis product treatment. In the present study, we asked whether TGFβ signaling was implicated in the induction of apoptosis by lipolysis products and whether there was a linkage between TGFβ receptor activation and pro-inflammatory responses. siRNA knockdown of ATF3 transcription abrogated both IL-8 and E-selectin up-regulation in lipolysis product-treated human aortic endothelial cells (HAEC). In addition, ATF3 knockdown prevented activation of caspase 3/7 and induction of p53, markers of apoptosis. Inhibition of the TGFβ Type I activin receptor-like kinase (ALK) receptors (ALKs 4, 5 and 7) by SB 431542 (10 μM) also inhibited inflammatory up-regulation, ATF3 induction and apoptosis. Both ATF3 knockdown and TGFβ receptor blockade prevented c-Jun phosphorylation, a co-activating transcription factor with ATF3 of AP-1 transcription sites. Our findings implicate TGFβ signaling in the inflammatory and apoptotic responses to TGRL lipolysis and suggest a complex interaction between TGFβ receptors and the SAPK subset of MAP Kinases.

The mTOR (Mammalian Target of Rapamycin) Kinase Maintains Integrity of mTOR Complex 2

In higher eukaryotes, growth factors promote anabolic processes and stimulate cell growth, proliferation, and survival by activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Deregulation of PI3K/Akt signaling is linked to human diseases, including cancer and metabolic disorders. The PI3K-dependent signaling kinase complex mTORC2 (mammalian target of rapamycin complex 2) has been defined as the regulatory Ser-473 kinase of Akt. The regulation of mTORC2 remains very poorly characterized. We have reconstituted mTORC2 by its assembly in vitro or by co-expression its four essential components (rictor, SIN1, mTOR, mLST8). We show that the functional mTOR kinase domain is required for the mTORC2 activity as the Ser-473 kinase of Akt. We also found that mTOR by phosphorylation of SIN1 prevents its lysosomal degradation. Thus, the kinase domain of mTOR is required for the functional activity of mTORC2, and it controls integrity of mTORC2 by maintaining the protein stability of SIN1.

Oleate prevents palmitate-induced cytotoxic stress in cardiac myocytes

The cytotoxicity of saturated fatty acids has been implicated in the pathophysiology of cardiovascular disease, though their effects on cardiac myocytes are incompletely understood. We examined the effects of palmitate and the mono-unsaturated fatty acid oleate on neonatal rat ventricular myocyte cell biology. Palmitate (0.5 mM) increased oxidative stress, as well as activation of the stress-associated protein kinases (SAPK) p38, Erk1/2, and JNK, following 18 h and induced apoptosis in ∼20% of cells after 24 h. Neither antioxidants nor SAPK inhibitors prevented palmitate-induced apoptosis. Low concentrations of oleate (0.1 mM) completely inhibited palmitate-induced oxidative stress, SAPK activation, and apoptosis. Increasing mitochondrial uptake of palmitate with l-carnitine decreased apoptosis, while decreasing uptake with the carnitine palmitoyl transferase-1 inhibitor perhexiline nearly doubled palmitate-induced apoptosis. These results support a model for palmitate-induced apoptosis, activation of SAPKs, and protein oxidative stress in myocytes that involves cytosolic accumulation of saturated fatty acids.

RXR α Stressed Out by MKK4

Stressful stimuli inhibit the activation of retinoids, but the mechanism of this inhibition has remained a mystery. Now Lee et al . have uncovered some of the molecular steps required for retinoid inhibition. Cells treated with anisomycin or expressing the constitutively activated stress-associated protein kinase kinase SEK1 (MKK4) showed reduced retinoic acid (RA)-mediated gene expression. Similarly treated cells contained phosphorylated retinoic X receptor (RXR) α but not phospho-RARα. MKK4 and JNK1 (Jun NH 2 -terminal kinase 1), the kinase downstream of MKK4 both phosphorylated RXRα, however, only phosphorylation of RXRα on Tyr 249 by MKK4 was essential for inhibiting RA-mediated transcription. Anisomycin-induced phosphorylation of RXRα was decreased in cells lacking MKK4. Thus, MKK4-directed RXRα phosphorylation may be important for attenuating RA responses in the presence of stress. Lee, H.-Y., Suh, Y.-A., Robinson, M.J., Clifford, J.L., Hong, W.K., Woodgett, J.R., Cobb, M.H., Mangelsdorf, D.J., and Kurie, J.M. (2000) Stress pathway activation induces phosphorylation of retinoid X receptor. J . Biol . Chem . 275 : 32193-32199. [Abstract] [Full Text]

Ras stimulates DNA topoisomerase II alpha through MEK: a link between oncogenic signaling and a therapeutic target.

Topoisomerase II alpha (topo II alpha) is a major target of antitumor treatments. In an effort to determine why this protein might be a better target in tumor cells than in normal cells, we attempted to determine if the altered proliferative signaling in a tumor cell might effect the levels of expression of the topo II alpha gene. In support of this idea, it was found that topo II alpha was elevated following microinjection of oncogenic Ras protein. Oncogenic ras was further shown to stimulate the topo II alpha promoter. Stimulation by ras was independent of the normal cell cycle regulation of this promoter. Transactivation of topo II alpha by ras required both the MEK/ERK pathway, and the stress-associated protein kinase (SAPK) signaling pathway. As a direct confirmation that both ERK and SAPK were involved in topo II alpha regulation, a constitutively active MEKK that stimulates these two kinases simultaneously was shown to strongly induce topo II alpha promoter activity. Activation of either pathway alone, on the other hand, only slightly stimulated the topo II alpha promoter. Deletion analyses showed that elements near both the 5' and 3' ends of the promoter were responsible for the ras stimulation. Site-directed mutagenesis further demonstrated that an Ets-like binding site near the 5' end (-480 to -475) was one of the responsive elements. Taken together, these studies demonstrate the direct role of Ras signaling in stimulation of topo II alpha expression, and thereby establish a link between the action of a common tumor mutation and the target of multiple anti-tumor reagents.

Growth hormone regulates ternary complex factors and serum response factor associated with the c-fos serum response element.

For insight into the mechanisms of gene regulation by growth hormone (GH), the regulation of transcription factors associated with the serum response element (SRE) located upstream of c-fos was examined. The SRE can mediate induction of reporter expression in response to GH. For insight into the mechanism by which GH regulates transcription factors, regulation of SRE-associated proteins by GH was examined. In nuclear extracts from 3T3-F442A fibroblasts, several SRE-binding complexes were identified by electrophoretic mobility shift assay. GH treatment for 2-10 min transiently increased binding of two complexes; binding returned to control values within 30 min. The two GH-stimulated complexes were supershifted by antibodies against the serum response factor (SRF), indicating that they contained SRF or an antigenically related protein. One of the GH-stimulated complexes was supershifted by antibody against Elk-1, suggesting that it contains a ternary complex factor (TCF) such as Elk-1 in addition to SRF. Induction of binding by GH was lost when the SRF binding site in the SRE was mutated, and mutation of either the SRF or TCF binding site altered the pattern of protein binding to the SRE. Mutation of the SRF or TCF binding site in SRE-luciferase plasmids inhibited the ability of GH to stimulate reporter expression, supporting a role for both SRF and TCF in GH-induced transcription of c-fos via the SRE. The TCF family member Elk-1 is capable of mediating GH-stimulated transcription, since GH-stimulated reporter expression was mediated by the transcriptional activation domain of Elk-1. Consistent with this stimulation, GH rapidly and transiently stimulated the serine phosphorylation of Elk-1. The increase was evident within 10 min and subsided after 30 min. Taken together, these data indicate that SRF and TCF contribute to GH-promoted transcription of c-fos via the SRE and are consistent with GH-promoted phosphorylation of Elk-1 contributing to GH-promoted transcriptional activation via the SRE.