Endogenous LXR signaling controls pulmonary surfactant homeostasis and prevents lung inflammation.

Lung type 2 pneumocytes (T2Ps) and alveolar macrophages (AMs) play crucial roles in the synthesis, recycling and catabolism of surfactant material, a lipid/protein fluid essential for respiratory function. The liver X receptors (LXR), LXRα and LXRß, are transcription factors important for lipid metabolism and inflammation. While LXR activation exerts anti-inflammatory actions in lung injury caused by lipopolysaccharide (LPS) and other inflammatory stimuli, the full extent of the endogenous LXR transcriptional activity in pulmonary homeostasis is incompletely understood. Here, using mice lacking LXRα and LXRß as experimental models, we describe how the loss of LXRs causes pulmonary lipidosis, pulmonary congestion, fibrosis and chronic inflammation due to defective de novo synthesis and recycling of surfactant material by T2Ps and defective phagocytosis and degradation of excess surfactant by AMs. LXR-deficient T2Ps display aberrant lamellar bodies and decreased expression of genes encoding for surfactant proteins and enzymes involved in cholesterol, fatty acids, and phospholipid metabolism. Moreover, LXR-deficient lungs accumulate foamy AMs with aberrant expression of cholesterol and phospholipid metabolism genes. Using a house dust mite aeroallergen-induced mouse model of asthma, we show that LXR-deficient mice exhibit a more pronounced airway reactivity to a methacholine challenge and greater pulmonary infiltration, indicating an altered physiology of LXR-deficient lungs. Moreover, pretreatment with LXR agonists ameliorated the airway reactivity in WT mice sensitized to house dust mite extracts, confirming that LXR plays an important role in lung physiology and suggesting that agonist pharmacology could be used to treat inflammatory lung diseases.

Reprogramming of the LXRα Transcriptome Sustains Macrophage Secondary Inflammatory Responses.

Macrophages regulate essential aspects of innate immunity against pathogens. In response to microbial components, macrophages activate primary and secondary inflammatory gene programs crucial for host defense. The liver X receptors (LXRα, LXRß) are ligand-dependent nuclear receptors that direct gene expression important for cholesterol metabolism and inflammation, but little is known about the individual roles of LXRα and LXRß in antimicrobial responses. Here, the results demonstrate that induction of LXRα transcription by prolonged exposure to lipopolysaccharide (LPS) supports inflammatory gene expression in macrophages. LXRα transcription is induced by NF-κB and type-I interferon downstream of TLR4 activation. Moreover, LPS triggers a reprogramming of the LXRα cistrome that promotes cytokine and chemokine gene expression through direct LXRα binding to DNA consensus sequences within cis-regulatory regions including enhancers. LXRα-deficient macrophages present fewer binding of p65 NF-κB and reduced histone H3K27 acetylation at enhancers of secondary inflammatory response genes. Mice lacking LXRα in the hematopoietic compartment show impaired responses to bacterial endotoxin in peritonitis models, exhibiting reduced neutrophil infiltration and decreased expansion and inflammatory activation of recruited F4/80lo-MHC-IIhi peritoneal macrophages. Together, these results uncover a previously unrecognized function for LXRα-dependent transcriptional cis-activation of secondary inflammatory gene expression in macrophages and the host response to microbial ligands.

Crosstalk Between LXR and Caveolin-1 Signaling Supports Cholesterol Efflux and Anti-Inflammatory Pathways in Macrophages.

Macrophages are immune cells that play crucial roles in host defense against pathogens by triggering their exceptional phagocytic and inflammatory functions. Macrophages that reside in healthy tissues also accomplish important tasks to preserve organ homeostasis, including lipid uptake/efflux or apoptotic-cell clearance. Both homeostatic and inflammatory functions of macrophages require the precise stability of lipid-rich microdomains located at the cell membrane for the initiation of downstream signaling cascades. Caveolin-1 (Cav-1) is the main protein responsible for the biogenesis of caveolae and plays an important role in vascular inflammation and atherosclerosis. The Liver X receptors (LXRs) are key transcription factors for cholesterol efflux and inflammatory gene responses in macrophages. Although the role of Cav-1 in cellular cholesterol homeostasis and vascular inflammation has been reported, the connection between LXR transcriptional activity and Cav-1 expression and function in macrophages has not been investigated. Here, using gain and loss of function approaches, we demonstrate that LXR-dependent transcriptional pathways modulate Cav-1 expression and compartmentation within the membrane during macrophage activation. As a result, Cav-1 participates in LXR-dependent cholesterol efflux and the control of inflammatory responses. Together, our data show modulation of the LXR-Cav-1 axis could be exploited to control exacerbated inflammation and cholesterol overload in the macrophage during the pathogenesis of lipid and immune disorders, such as atherosclerosis.

Synthesis and Antiplasmodial Activity of 1,2,3-Triazole-Naphthoquinone Conjugates.

A series of 34 1,2,3-triazole-naphthoquinone conjugates were synthesized via copper-catalyzed cycloaddition (CuAAC). They were evaluated for their in vitro antimalarial activity against chloroquine-sensitive strains of Plasmodium falciparum and against three different tumor cell lines (SKBr-3, MCF-7, HEL). The most active antimalarial compounds showed a low antiproliferative activity. Simplified analogues were also obtained and some structure-activity relationships were outlined. The best activity was obtained by compounds 3s and 3j, having IC50 of 0.8 and 1.2 µM, respectively. Molecular dockings were also carried on Plasmodium falciparum enzyme dihydroorotate dehydrogenase (PfDHODH) in order to rationalize the results.

Signal transducer and activator of transcription (STAT)-5: an opportunity for drug development in oncohematology.

The signal transducer and activator of transcription (STAT) are transcription factors that work via JAK/STAT pathway regulating the expression of genes involved in cell survival, proliferation, differentiation, development, immune response, and, among other essential biological functions, hematopoiesis. JAK/STAT signaling is strictly regulated under normal physiological conditions. However, a large group of diverse diseases has been associated to an aberrant regulation of STAT factors. Erroneous modulation of the pathway leads to constitutive STAT activation, thereby driving proliferation, inflammation, and an uncontrolled immune response. Deregulated STAT5 activation has been found in the development of many hematopoietic tumors, including chronic and acute leukemias, polycythemia vera, and lymphoma. Mutations in the kinases that phosphorylate STAT5, and/or overexpression of the upstream receptor-associated tyrosine kinases have been suggested as the main drivers of constitutive STAT5 activation. Hyper-activated STAT5 leads to the aberrant expression of its target genes including antiapoptotic, proliferative, and pro-inflammatory genes, favouring tumorigenesis. In this review, we intent to discuss the biology of JAK/STAT pathway, with particular focus on STAT5 and its crucial role in the development and progression of hematologic malignancies. Furthermore, we provide a synopsis of potential therapeutic strategies based on STAT5 activity inhibition that may represent an excellent opportunity for drug development in oncohematology.

Correction: CM363, a novel naphthoquinone derivative which acts as multikinase modulator and overcomes imatinib resistance in chronic myelogenous leukemia.

[This corrects the article DOI: 10.18632/oncotarget.11425.].

Lawsone, Juglone, and ß-Lapachone Derivatives with Enhanced Mitochondrial-Based Toxicity.

Naphthoquinones are among the most active natural products obtained from plants and microorganisms. Naphthoquinones exert their biological activities through pleiotropic mechanisms that include reactivity against cell nucleophiles, generation of reactive oxygen species (ROS), and inhibition of proteins. Here, we report a mechanistic antiproliferative study performed in the yeast Saccharomyces cerevisiae for several derivatives of three important natural naphthoquinones: lawsone, juglone, and ß-lapachone. We have found that (i) the free hydroxyl group of lawsone and juglone modulates toxicity; (ii) lawsone and juglone derivatives differ in their mechanisms of action, with ROS generation being more important for the former; and (iii) a subset of derivatives possess the capability to disrupt mitochondrial function, with ß-lapachones being the most potent compounds in this respect. In addition, we have cross-compared yeast results with antibacterial and antitumor activities. We discuss the relationship between the mechanistic findings, the antiproliferative activities, and the physicochemical properties of the naphthoquinones.

A Novel Naphthoquinone-Coumarin Hybrid That Inhibits BCR-ABL1-STAT5 Oncogenic Pathway and Reduces Survival in Imatinib-Resistant Chronic Myelogenous Leukemia Cells.

BCR-ABL1-STAT5 is an oncogenic signaling pathway in human chronic myelogenous leukemia (CML) and it represents a valid target for anti-CML drug design. Resistance to direct BCR-ABL1 inhibitors is a common clinical issue, so STAT5 inhibition has become an interesting alternative target. In this study, the effects of NPQ-C6, a novel naphtoquinone-coumarin conjugate, were evaluated on human CML-derived K562 cells. Live-Cell Imaging analysis revealed that NPQ-C6 inhibited 2D (IC50AUC = 1.4 ± 0.6 µM) growth of CML cells. NPQ-C6 increased sub-G1 and reduced G0/G1 cell cycle phases in a dose- and time-dependent manner. This effect on cell cycle was related to increased levels of apoptotic nuclei, cleavage of caspase-3, -9, and PARP and annexin V-positive cells. NPQ-C6 increased γH2AX, a double-strand DNA break marker. NPQ-C6 showed a wide range of modulatory effects on cell signaling through an early increased phosphorylation of JNK, P38-MAPK and AKT, and decreased phosphorylation of ERK1/2, BCR-ABL1, and STAT5. NPQ-C6 inhibited expression of c-MYC and PYM-1, two target gene products of BCR-ABL1/STAT5 signaling pathway. Cytokine-induced activation of STAT5/STAT3-dependent transcriptional and DNA binding activities were also inhibited by NPQ-C6. Notably, NPQ-C6 maintained its activity on BCR-ABL1/STAT5/c-MYC/PIM-1 oncogenic pathway in imatinib-resistant cells. Molecular modeling suggested BCR-ABL1 and JAK2 proteins as NPQ-C6 targets. In summary, our data show a novel multikinase modulator that might be therapeutically effective in BCR-ABL1-STAT5-related malignancies.

In vitro activity of 1H-phenalen-1-one derivatives against Acanthamoeba castellanii Neff and their mechanisms of cell death.

Acanthamoeba is an opportunistic pathogen which is the causal agent of a sight-threatening ulceration of the cornea known as Acanthamoeba keratitis (AK) and, more rarely, an infection of the central nervous system called "granulomatous amoebic encephalitis" (GAE). The symptoms of AK are non-specific, and so it can be misdiagnosed as a viral, bacterial, or fungal keratitis. Furthermore, current therapeutic measures against AK are arduous, and show limited efficacy against the cyst stage of Acanthamoeba. 1H-Phenalen-1-one (PH) containing compounds have been isolated from plants and fungi, where they play a crucial role in the defense mechanism of plants. Natural as well as synthetic PHs exhibit a diverse range of biological activities against fungi, protozoan parasites or human cancer cells. New synthetic PHs have been tested in this study and they show a potential activity against this protozoa.

5-Ethynylarylnaphthalimides as antitumor agents: Synthesis and biological evaluation.

A set of 5-ethynylarylnaphthalimides was synthesized by Sonogashira cross-coupling reactions and evaluated for antiproliferative and antitopoisomerase II in vitro activities. Furthermore docking studies of these molecules as DNA-intercalators were carried out and the in vivo DNA-damaging activity was also determined with the model organism Saccharomyces cerevisiae. From the obtained results three naphthalimides 6, 13 and 14 showed strong topoisomerase II inhibitory activity. These three molecules also presented good docking scores as DNA-intercalators using a self-complementary oligodeoxynucleotide d(ATGCAT)2 as a model, and compounds 13 and 14 were among the most cytotoxic in the in vivo DNA-damaging activity.

CM363, a novel naphthoquinone derivative which acts as multikinase modulator and overcomes imatinib resistance in chronic myelogenous leukemia.

Human Chronic Myelogenous Leukemia (CML) is a hematological stem cell disorder which is associated with activation of Bcr-Abl-Stat5 oncogenic pathway. Direct Bcr-Abl inhibitors are initially successful for the treatment of CML but over time many patients develop drug resistance. In the present study, the effects of CM363, a novel naphthoquinone (NPQ) derivative, were evaluated on human CML-derived K562 cells. CM363 revealed an effective cell growth inhibition (IC50 = 0.7 ± 0.5 µM) by inducing cancer cells to undergo cell cycle arrest and apoptosis. CM363 caused a dose- and time-dependent reduction of cells in G0/G1 and G2/M phases. This cell cycle arrest was associated with increased levels of cyclin E, pChk1 and pChk2 whereas CM363 downregulated cyclin B, cyclin D3, p27, pRB, Wee1, and BUBR1. CM363 increased the double-strand DNA break marker γH2AX. CM363 caused a time-dependent increase of annexin V-positive cells, DNA fragmentation and increased number of apoptotic nuclei. CM363 triggered the mitochondrial apoptotic pathway as reflected by a release of cytochrome C from mitochondria and induction of the cleavage of caspase-3 and -9, and PARP. CM363 showed multikinase modulatory effects through an early increased JNK phosphorylation followed by inhibition of pY-Bcrl-Abl and pY-Stat5. CM363 worked synergistically with imatinib to inhibit cell viability and maintained its activity in imatinib-resistant cells. Finally, CM363 (10 mg/Kg) suppressed the growth of K562 xenograft tumors in athymic mice. In summary, CM363 is a novel multikinase modulator that offers advantages to circumvent imanitib resistance and might be therapeutically effective in Bcrl-Abl-Stat5 related malignancies.

Synthesis and study of antiproliferative, antitopoisomerase II, DNA-intercalating and DNA-damaging activities of arylnaphthalimides.

A series of arylnaphthalimides were designed and synthesized to overcome the dose-limiting cytotoxicity of N-acetylated metabolites arising from amonafide, the prototypical antitumour naphthalimide whose biomedical properties have been related to its ability to intercalate the DNA and poison the enzyme Topoisomerase II. Thus, these arylnaphthalimides were first evaluated for their antiproliferative activity against two tumour cell lines and for their antitopoisomerase II in vitro activities, together with their ability to intercalate the DNA in vitro and also through docking modelization. Then, the well-known DNA damage response in Saccharomyces cerevisiae was employed to critically evaluate whether these novel compounds can damage the DNA in vivo. By performing all these assays we conclude that the 5-arylsubstituted naphthalimides not only keep but also improve amonafide's biological activities.

Synthesis and in vitro antiprotozoal evaluation of substituted phenalenone analogues.

A set of derivatives encompassing structural modifications on the privileged phenalenone scaffold were assessed for their antiparasitic activities against the most clinically relevant forms of trypanosomiasis and leishmaniasis. Several compounds exhibited leishmanicidal effects at levels comparable or better than the reference drug pentamidine, while the parent phenalenone was shown to have a level of activity against Trypanosoma cruzi comparable to the marketed drug benznidazole.