322,915 research outputs found
JNK signalling in cancer: In need of new, smarter therapeutic targets
No full textCopyright © 2013 The British Pharmacological Society. This is the accepted version of the following article: Bubici, C. and Papa, S. (2014), JNK signalling in cancer: in need of new, smarter therapeutic targets. British Journal of Pharmacology, 171: 24–37, which has been published in final form at http://onlinelibrary.wiley.com/doi/10.1111/bph.12432/abstract.The JNKs are master protein kinases that regulate many physiological processes, including inflammatory responses, morphogenesis, cell proliferation, differentiation, survival and death. It is increasingly apparent that persistent activation of JNKs is involved in cancer development and progression. Therefore, JNKs represent attractive targets for therapeutic intervention with small molecule kinase inhibitors. However, evidence supportive of a tumour suppressor role for the JNK proteins has also been documented. Recent studies showed that the two major JNK proteins, JNK1 and JNK2, have distinct or even opposing functions in different types of cancer. As such, close consideration of which JNK proteins are beneficial targets and, more importantly, what effect small molecule inhibitors of JNKs have on physiological processes, are essential. A number of ATP-competitive and ATP-non-competitive JNK inhibitors have been developed, but have several limitations such as a lack of specificity and cellular toxicity. In this review, we summarize the accumulating evidence supporting a role for the JNK proteins in the pathogenesis of different solid and haematological malignancies, and discuss many challenges and scientific opportunities in the targeting of JNKs in cancer.Kay Kendall Leukemia Fund,
Italian Association for Cancer Research and Foundation for Liver Research
Screening Kinase-Dependent Phosphorylation of Key Metabolic Reprogramming Regulators
No full textAerobic glycolysis has been commonly linked to cell proliferation, especially in cancer cells where it serves to generate sufficient energy and biosynthesis of new cell constituents needed for cell growth and division. The M2 isoform of pyruvate kinase (PKM2) catalyzes the last reaction of the glycolytic process. PKM2 promotes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to ADP, generating ATP and releasing pyruvate. This rate-limiting reaction relies therefore on the enzymatic activity of PKM2. The switching between the high- and low-activity states of PKM2 is subjected to a combination of allosteric mechanisms and fine-tuned regulation by oncogenes and tumor suppressor genes. These regulatory mechanisms involve primarily post-translational modifications of PKM2. Recent findings suggest that phosphorylation contributes to the regulation of PKM2 activity.
Here, we describe an in vitro kinase assay we used to assess PKM2 phosphorylation by c-Jun N-terminal kinase (JNK), a master regulator of apoptosis, cell proliferation, and differentiation. While the use of phospho-specific antibodies gives information in terms of measuring the effects of a given kinase on its substrate, specific antibodies for newly identified phospho-groups are not readily available. The in vitro kinase assay allows the immediate measuring of phosphorylation of any substrate of interest. Although there are several options that do not use radioactive materials, we continue to rely on this biochemical method for robust quantitation of results. More interestingly, this protocol can be easily adapted to measure the activity of other kinases by using their specific substrates
An Integrated Methodology to Quantify the Glycolytic Stress in Plasma Cell Myeloma in Response to Cytotoxic Drugs
No full textMultiple myeloma (MM) is an incurable plasma cell malignancy primarily localized within the bone marrow (BM). Myeloma plasma cells, like many other cancer cells, change their metabolism in response to internal and external stimuli. The main metabolic alterations of MM cells include deregulated glycolysis (commonly associated with enhanced uptake and utilization of glucose), lipid metabolism dysregulation, as well as deregulated mitochondrial respiration (commonly associated with the deregulated formation of reactive oxygen species). Over the past decade, the discovery of novel methodologies and the commercialization of sophisticated instrumentation and reagents have facilitated the detection of real-time changes in cellular bioenergetics. Of those, the Seahorse™ extracellular flux (XF) analyzer has been widely used to evaluate the glycolytic flux and mitochondrial respiration in many cell types. While adherent cell lines are easy to use with this technology, non-adherent suspension cells are more difficult to handle especially when their metabolic activities are being investigated in response to drug treatment. Here, we provide an integrated protocol that allows the detection of extracellular acidification rate (ECAR) of live myeloma plasma cells in response to chemotherapeutic drugs. Our optimized protocol consists of treating myeloma cells with cytotoxic drug of interest in a standard culture plate prior to the real-time analysis in the XF analyzer. Furthermore, we provide results of experiments in which the metabolic activities of myeloma cells in response to cytotoxic treatment were compared between the manufacturer’s basic procedure and our optimized protocol. Our observations suggest that our integrated protocol can be used to achieve consistent, well-standardized results and thus it may have broad applications in studies focusing on the characterization of metabolic events in non-adherent suspension cells
Mechanisms of liver disease: cross-talk between the NF-kappa B and JNK pathways
No full textThe liver plays a central role in the transformation anddegradation of endogenous and exogenous chemicals,and in the removal of unwanted cells such as damaged,genetically mutated and virus-infected cells. Because ofthis function, the liver is susceptible to toxicity causedby the products generated during these natural occur-rences. Hepatocyte death is the major feature of liverinjury. In response to liver injury, specific intracellularprocesses are initiated to maintain liver integrity. Inflam-matory cytokines including tumor necrosis factor (TNF)aand interleukin-6 (IL-6) are key mediators of these pro-cesses and activate different cellular response such asproliferation, survival and death. TNFainduces specificsignaling pathways in hepatocytes that lead to activationof either pro-survival mediators or effectors of cell death.Whereas activation of transcription factor NF-kBpro-motes survival, c-Jun N-terminal kinases (JNKs) andcaspases are strategic effectors of cell death in theTNFa-mediated signaling pathway. This review summa-rizes recent advances in the mechanisms of TNFa-induced hepatotoxicity and suggests that NF-kB plays aprotective role against JNK-induced hepatocyte death.Identification of the mechanisms regulating interplaybetween the NF-kB and JNK pathways is required in thesearch for novel targets for the treatment of liver disease,including hepatitis and hepatocellular carcinoma
In the Crosshairs: NF-κB Targets the JNK Signaling Cascade.
No full textNF-κB/Rel transcription factors are well-known for their roles in the regulation of inflammation and immunity. NF-κB also blocks programmed cell death (PCD) or apoptosis triggered by proinflammatory cytokine, tumor necrosis factor (TNF)α. Through transcriptional induction of distinct subsets of cyto-protective target genes, NF-κB inhibits the execution of apoptosis activated by this cytokine. This protective action is mediated, in part, by factors (such as A20, GADD45β, and XIAP) that downregulate the pro-apoptotic c-Jun-N-terminal (JNK) pathway. A suppression of reactive oxygen species (ROS), which are themselves major cell death-inducing elements activated by TNFα, is an additional protective function recently ascribed to NF-κB. This function of NF-κB involves an induction of mitochondrial anti-oxidant enzyme, manganese superoxide dismutase (Mn-SOD), and a control of cellular iron availability through upregulation of Ferritin heavy chain – one of two subunits of Ferritin, the major iron storage protein complex of the cell. An emerging view of NF-κB is that, while integrated, its actions in immunity and in promoting cell survival are executed through upregulation of distinct subsets of target genes. Thus, these inducible blockers of apoptosis may provide potential new targets to inhibit specific functions of NF-κB. In the future, this might allow for a better treatment of complex human diseases involving dysregulated NF-κB activity, including chronic inflammatory conditions and cancer
The Codex Major of the Collectio Altaempsiana: a Non-invasive NMR Study of Paper
No full textA new portable nuclear magnetic resonance (NMR) device allows in situ non-invasive investigation of paper in order to examine some
aspects of the microscopic healthy state of documents of historical and artistic interest. The apparatus has aNMRsurface probe, which permits
to perform most of the NMR relaxometric measurements on objects of almost every size and shape. It uses non-ionizing radio frequency
electromagnetic waves and is easily transportable without hazard for people or environment. Some results obtained with this device on the
pages of the Codex Major, a manuscript musical anthology that belongs to the Collectio Altaempsiana (1600–1610) of Palazzo Altaemps in
Rome, are here presented. The NMR results provide indications about the spread of the deterioration process of the paper and of the corrosion
effect caused by the iron-gall ink
The NF-κB Transcription Factor Pathway as a Therapeutic Target in Cancer: Methods for Detection of NF-κB Activity
No full textNF-kappaB transcription factors marshal innate and adaptive immunity and inflammation. NF-kappaB also counters programmed cell death (PCD) induced by the proinflammatory cytokine tumor necrosis factor (TNF)alpha, and this activity of NF-kappaB is crucial for organismal physiology, chronic inflammation, and tumorigenesis. Indeed, whereas NF-kappaB contributes to many aspects of oncogenesis, it is now clear that its suppressive action on PCD is central to this process. Notably, recent studies indicate that NF-kappaB represents a crucial link in the well-established association between inflammation and carcinogenesis. In this link, NF-kappaB promotes synthesis of inflammatory mediators (e.g. TNFalpha) that stimulate growth of cancer cells, and upregulates genes that protect these cells against PCD induced by inflammatory signals. Elevated NF-kappaB activity also hampers tumor-cell killing inflicted by radiation and chemotherapeutic drugs, and in so doing, promotes resistance to anticancer therapy. Accordingly, NF-kappaB-targeting drugs are increasingly being used for treatment of human malignancies. Owing to the ubiquitous nature of the NF-kappaB pathway, however, these drugs have serious side effects, which limit their clinical use. Thus, a preferable approach would be to block, rather than NF-kappaB itself, its critical downstream targets that mediate discrete functions in cancer, such as prosurvival functions. Recent discoveries unraveling tissue specificity in the NF-kappaB-inducible mechanism(s) for control of PCD and identifying putative effectors of this control clearly validate this therapeutic approach. Given the emerging role of TNFkappa-induced signals of NF-kappaB activation in cancer and the potential of these signals for yielding new anticancer therapies, we focus herein on the methods most commonly used for analysis of the molecular steps leading from the triggering of TNF-Receptor (TNF-R)1 - the primary receptor of TNFalpha - to the induction of NF-kappaB. Specifically, we review the methods used for analysis of TNF-R1 trafficking, assembly of so-called TNF-R1 complex I, formation and activation of the IkappaB kinase (IKK) complex, phosphorylation and proteolysis of inhibitory IkappaB proteins, post-translational modifications and nuclear translocation of NF-kappaB dimers, induction of NF-kappaB transcriptional activity and binding to specific promoters, and upregulation of NF-kappaB target genes. The analysis of these events in cancerous cells may not only provide a better understanding of the basis for the role of NF-kappaB in carcinogenesis, but also potential new targets for selective anticancer therapy
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