NFκB signaling in carcinogenesis and as a potential molecular target for cancer therapy

It has become increasingly clear that deregulation of the NFκB signaling cascade is a common underlying feature of many human ailments including cancers. The past two decades of intensive research on NFκB has identified the basic mechanisms that govern the functioning of this pathway but uncovering the details of why this pathway works differently in different cellular contexts or how it interacts with other signaling pathways remains a challenge. A thorough understanding of these processes is needed to design better and more efficient therapeutic approaches to treat complex diseases like cancer. In this review, we summarize the literature documenting the involvement of NFκB in cancer, and then focus on the approaches that are being undertaken to develop NFκB inhibitors towards treatment of human cancers.     

Autophagy is required for the activation of NFκB

It is well-established that the activation of the inhibitor of NFκB (IκBα) kinase (IKK) complex is required for autophagy induction by multiple stimuli. Here, we show that in autophagy-competent mouse embryonic fibroblasts (MEFs), distinct autophagic triggers, including starvation, mTOR inhibition with rapamycin and p53 inhibition with cyclic pifithrin α lead to the activation of IKK, followed by the phosphorylation-dependent degradation of IκBα and nuclear translocation of NFκB. Remarkably, the NFκB signaling pathway was blocked in MEFs lacking either the essential autophagy genes Atg5 or Atg7. In addition, we found that tumor necrosis factor α (TNFα)-induced NFκB nuclear translocation is abolished in both Atg5- and Atg7-deficient MEFs. Similarly, the depletion of essential autophagy modulators, including ATG5, ATG7, Beclin 1 and VPS34, by RNA interference inhibited TNFα-driven NFκB activation in two human cancer cell lines. In conclusion, it appears that, at least in some instances, autophagy is required for NFκB activation, highlighting an intimate crosstalk between these two stress response signaling pathways.     

Expression of cell kinetics and death during monocyte–macrophage differentiation: effects of Actinomycin D and Vinblastine treatments

The different effects of two cytostatic drugs, Actinomycin D and Vinblastine, during macrophage-like differentiation induced in THP-1 monocytic cell line by phorbol ester phorbol 12-myristate 13-acetate (PMA) (6, 30, and 60 nM), were studied by morpho-cytochemical approaches. In PMA-unstimulated monocytic cells, the cytostatic effects of Actinomycin D (an antimetabolic drug) were characterized by a drastic reduction of the G2/M cells accompanied by dramatic death of the G1 cells; on the contrary, Vinblastine (a microtubule-depolymerizating drug) induced an accumulation of the G2/M cells with the appearance of aneugenic micronuclei and scarce cell death mainly from the G1 cells. After 60 nM PMA stimulation, the culture was mostly composed by macrophagic cells characterized by low proliferation and the appearance of mono-/binucleated polyploid cells; in this condition, the cytotoxicity of the two drugs, more effective for Vinblastine, induced cell death in the different ploidy classes (2c, 4c, 8c). Cell death appeared to be of apoptotic nature, but with some morpho-phenotypic differences due to the action mechanism of the drugs and dependent on cell culture growth and differentiation. As a consequence of the different block-action of the two drugs on the cell cycle phases and in relation to the different subcellular targets, the effects changed during the transition from not-adhering/proliferating monocytes to adhering/low-proliferating differentiated macrophages.     

Copper is a potent inhibitor of both the canonical and non-canonical NFκB pathways

Copper is an essential trace element that plays key roles in many metabolic processes. Homeostatic regulation of intracellular copper is normally tightly controlled, but deregulated copper levels are found in numerous metabolic and neurodegenerative diseases, as well as in a range of neoplasms. There are conflicting reports regarding the exact role of copper in the regulation of NFκB-responsive genes, specifically whether copper leads to increased activation of the NFκB pathways, or downregulation. Here we show that increased intracellular levels of copper, using the ionophore clioquinol, leads to a potent inhibition of NFκB pathways, induced by multiple distinct stimuli. Addition of copper to cells inhibits ubiquitin-mediated degradation of IκBα by preventing its phoshorylation by the upstream IKK complex. Intriguingly, copper-dependent inhibition of NFκB can be reversed by the addition of the reducing agent, N-acetylcysteine (NAC). These results suggest that the oxidative properties of excess copper prevent NFκB activation by blocking IκBα destruction, and that NFκB activity should be assessed in diseases associated with copper excess.     

Interactions between NFκB and Its Inhibitor iκB: Biophysical Characterization of a NFκB/iκB-α Complex

The N-terminal domain (1–318 amino acids) of mouse NFB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length iB- (MAD3, 1–317 amino acids) molecule was generated by binding the E. coli-derived iB- to the purified NFB and purifying the complex by sequential chromatography. The stoichiometry of NFB to iB in the complex was determined to be 2 to 1 by light scattering and SDS–polyacrylamide gel electrophoresis. The secondary structure of the NFB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFB occurs upon binding of DNA. The FTIR spectrum of the NFB/iB complex indicates that its secondary structure is composed of 17% -helix, 39% -strand, 18% irregular structures, and 26% -turns and loops. By comparing these data to the FTIR data for NFB alone, it is concluded that the iB (MAD3) in the complex contains 35% -helix, 27% -strand, 22% irregular structures, and 16% -turns and loops. Circular dichroism (CD) analysis of a shorter form of iB (pp40) indicates that it contains at least 20% -helix and that the iB subunit accounts for nearly all of the -helix present in the NFB/iB complex, consistent with the FTIR results. The stabilities of NFB, iB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of iB is enhanced upon the formation of the NFB/iB complex.     

Micro-RNAs and macrophage diversity in atherosclerosis: New players,new challenges…new opportunities for therapeutic intervention?

Efforts in experimental therapeutics of atherosclerosis are mostly focused on identifying candidate targets that can be exploited in developing new strategies to reduce plaque progression, induce its regression and/or improve stability of advanced lesions. Plaque macrophages are central players in all these processes, and consequently a significant amount of research is devoted to understanding mechanisms that regulate, for instance, macrophage apoptosis, necrosis or migration. Macrophage diversity is a key feature of the macrophage population in the plaque and can impact many aspects of lesion development. Thus, searching for molecular entities that contribute to atherorelevant functions of a specific macrophage type but not others may lead to identification of targets that can be exploited in phenotype selective modulation of the lesional macrophage. This however, remains an unmet goal. In recent years several studies have revealed critical functions of micro-RNAs (miRs) in mechanisms of macrophage polarization, and a number of miRs have emerged as being specific of distinctive macrophage subsets. Not only can these miRs represent the first step towards recognition of phenotype specific targets, but they may also pave the way to reveal novel atherorelevant pathways within macrophage subsets. This article discusses some of these recent findings, speculates on their potential relevance to atherosclerosis and elaborates on the prospective use of miRs to affect the function of plaque macrophages in a phenotype selective manner.     

Glucocorticoid-mediated anti-inflammatory effect through NFκB is preserved in the absence of Dexras1

Glucocorticoids effectively mediate the resolution of inflammation, but long-term use of glucocorticoids inevitably causes metabolic side effects. However, it is unknown if metabolic effectors such as Dexras1, a dexamethasone-stimulated protein, play a role in the anti-inflammatory outcome of dexamethasone. Here, we demonstrate that Dexras1 is required for the dexamethasone-induced upregulation of annexin A1 expression, but is not involved in the reduction of inflammation as evidenced by decreased pro-inflammatory parameters. In the absence of Dexras1, lipopolysaccharide (LPS)-induced interleukin-6 expression was suppressed when murine macrophage RAW264.7 cells were treated with dexamethasone. Similar observations were made in the blood of Dexras1 knockout mice. Furthermore, dexamethasone suppressed the LPS-stimulated increase of NFκB-p65 in both control and Dexras1-absent RAW264.7 cells. Interestingly, depletion of Dexras1 resulted in the loss of pERK production. These results suggest that Dexras1 is involved primarily in the metabolic side effects and its inhibition preserves the anti-inflammatory action of glucocorticoids. Thus, the inhibition of Dexras1 will be an excellent target for reducing steroid-induced side effects.