Suppression of Proliferation of Human Glioblastoma Cells by Combined Phosphodiesterase and Multidrug Resistance-Associated Protein 1 Inhibition

The paucity of currently available therapies for glioblastoma multiforme requires novel approaches to the treatment of this brain tumour. Disrupting cyclic nucleotide-signalling through phosphodiesterase (PDE) inhibition may be a promising way of suppressing glioblastoma growth. Here, we examined the effects of 28 PDE inhibitors, covering all the major PDE classes, on the proliferation of the human U87MG, A172 and T98G glioblastoma cells. The PDE10A inhibitors PF-2545920, PQ10 and papaverine, the PDE3/4 inhibitor trequinsin and the putative PDE5 inhibitor MY-5445 potently decreased glioblastoma cell proliferation. The synergistic suppression of glioblastoma cell proliferation was achieved by combining PF-2545920 and MY-5445. Furthermore, a co-incubation with drugs that block the activity of the multidrug resistance-associated protein 1 (MRP1) augmented these effects. In particular, a combination comprising the MRP1 inhibitor reversan, PF-2545920 and MY-5445, all at low micromolar concentrations, afforded nearly complete inhibition of glioblastoma cell growth. Thus, the potent suppression of glioblastoma cell viability may be achieved by combining MRP1 inhibitors with PDE inhibitors at a lower toxicity than that of the standard chemotherapeutic agents, thereby providing a new combination therapy for this challenging malignancy.

Naringenin Enhances Anti-Proliferation Effect of FMSP On K562 Human Chronic Myelogenous Leukemia Cells Via Targeting Calmodulin Signaling Pathway

Background: FMSP is a synthesized ferrocene derivative which possesses strong anti-proliferative and apoptosis inducing characteristics on tumor cells. Naringenin as a polyphenolic flavonoid is also able to reduce cell viability in tumors.Methods: Cell viability and proliferation of cancer cells after treatment with these agents were determined with MTT assay. To predict the possible interaction between calmodulin (CaM) and FMSP and naringenin, docking stuies were performed. By using fluorescence emission spectra, effects of FMSP and naringenin on CaM structure and activity were studied. CaM-dependent activation of phosphodiesterase 1 (PDE1) by FMSP in relation to naringenin and their combination were compared. Effects of these compounds on PDE1 inhibition, cAMP accumulation, and cAMP-dependent protein kinase A (PKA) activation were assayed. Results: Our results showed that combination of FMSP and naringenin had more inhibitory effects on CaM structure than FMSP and naringenin alone. Results of docking analyses also confirmed efficient intraction of the two compunds with hydrophobic pocket of calmodulin active site. Kinetic analyses of these agents interaction with CaM showed FMSP and naringenin both competitively inhibited PDE1 activation without changing Vmax parameter. FMSP and naringenin synergistically increased Km values in a higher level compared to FMSP or naringenin alone. Combination of these two agents also had more cytotoxic effects on K562 cells than FMSP alone. Conclusions: It was shown that mechanism of K562 cell proliferation inhibition by these compounds is based on CaM and consequent PDE inhibition followed by intracellular cAMP level elevation and increased PKA activity in a dose-dependent manner.

Neuroinflammation in Ischemic Stroke: Inhibition of cAMP-Specific Phosphodiesterases (PDEs) to the Rescue

Ischemic stroke is caused by a thromboembolic occlusion of a major cerebral artery, with the impaired blood flow triggering neuroinflammation and subsequent neuronal damage. Both the innate immune system (e.g., neutrophils, monocytes/macrophages) in the acute ischemic stroke phase and the adaptive immune system (e.g., T cells, B cells) in the chronic phase contribute to this neuroinflammatory process. Considering that the available therapeutic strategies are insufficiently successful, there is an urgent need for novel treatment options. It has been shown that increasing cAMP levels lowers neuroinflammation. By inhibiting cAMP-specific phosphodiesterases (PDEs), i.e., PDE4, 7, and 8, neuroinflammation can be tempered through elevating cAMP levels and, thereby, this can induce an improved functional recovery. This review discusses recent preclinical findings, clinical implications, and future perspectives of cAMP-specific PDE inhibition as a novel research interest for the treatment of ischemic stroke. In particular, PDE4 inhibition has been extensively studied, and is promising for the treatment of acute neuroinflammation following a stroke, whereas PDE7 and 8 inhibition more target the T cell component. In addition, more targeted PDE4 gene inhibition, or combined PDE4 and PDE7 or 8 inhibition, requires more extensive research.

Possible Tracheal Relaxant and Antimicrobial Effects of the Essential Oil of Ethiopian Thyme Species (Thymus serrulatus Hochst. ex Benth.): A Multiple Mechanistic Approach

The genus Thymus is traditionally used for the treatment of hyperactive airways complaints. The purpose of the current study is to investigate the potential tracheal relaxant effect and possible mechanism(s) of the essential oil of Thymus serrulatus (TS Oil) in isolated guinea pig tracheal tissues. The essential oil was obtained from the fresh erial parts of Thymus serrulatus, and its phyto-components were identified by GC-MS analysis. Guinea pig tracheal preparations were used for testing the tracheal relaxant effect of TS Oil with the determination of the mechanism(s) involved in this relaxation. GC-MS findings reveal that terpenes, fragrance constituents, saponins, and higher fatty acids are present in TS Oil. In isolated guinea pig trachea, TS Oil inhibited carbachol (CCh, 1 µM) and K+ (80 mM)-induced contractions in a pattern similar to that of dicyclomine. TS Oil, at 0.3 mg/ml, shifted parallel CCh-curves towards the right, followed by a non-parallel shift at higher concentration (1 mg/ml), thus suppressing maximum response in the same manner as produced by dicyclomine. Pretreatment of tissues with TS Oil (1 and 3 mg/ml) also produced a rightward shift of Ca++ concentration-response curves (CRCs) in the same manner as caused by verapamil. Further, TS Oil at low concentrations (0.3 and 1 mg/ml) shifted isoprenaline-induced inhibitory CRCs towards the left and increased cAMP levels in isolated tracheal homogenates similar to papaverine, a phosphodiesterase (PDE) inhibitor. In the antimicrobial assay performed by the agar well diffusion method, TS Oil was found most active against Candida albicans and Staphylococcus aureus where the zone of inhibition measured was 28 mm. Additionally, there was little difference between standard strains of gram-positive and gram-negative bacteria. However, methicillin-resistant S. aureus (MRSA) showed a small zone of inhibition as compared to standard strains (22 mm). From these results, it can be concluded that the essential oil of T. serrulatus has the potential to produce antimicrobial effects while causing tracheal relaxation mediated possibly by anticholinergic effects, Ca++ channel blockade, and PDE inhibition whereas additional mechanism(s) cannot be ruled out.

A metabolomics insight into the Cyclic Nucleotide Monophosphate signaling cascade in tomato under non-stress and salinity conditions

ABSTRACTCyclic Nucleotides Monophosphate (cNMP) are key signalling compounds whose role in plant cell signal transduction is till poorly understood. In this work we used sildenafil, a phosphodiesterase (PDE) inhibitor used in human, to amplify the signal cascade triggered by cNMP using tomato as model plant. Metabolomics was then used, together with plant growth and root architecture parameters, to unravel the changes elicited by PDE inhibition either under non-stress and 100 mM NaCl salinity conditions.The PDE inhibitor elicited a significant increase in biomass (+62%) and root length (+56%) under no stress conditions, and affected root architecture in terms of distribution over diameter classes. Together with cGMP, others cNMP were modulated by the treatment. Moreover, PDE inhibition triggered a broad metabolic reprogramming involving photosynthesis and secondary metabolism. A complex crosstalk network of phytohormones and other signalling compounds could be observed in treated plants. Nonetheless, metabolites related to redox imbalance processes and NO signalling could be highlighted in tomato following PDE application. Despite salinity damped down the growth-promoting effects of sildenafil, interesting implications in plant mitigation to stress-related detrimental effects could be observed.HIGHLIGHTThe role of Cyclic Nucleotides Monophosphate in plant cell signal transduction involves regulation of plant growth and architecture, together with a broad biochemical reprogramming of metabolism.

Cell dispersal by localized degradation of a chemoattractant

Chemotaxis, the guided motion of cells by chemical gradients, plays a crucial role in many biological processes. In the social amoeba Dictyostelium discoideum, chemotaxis is critical for the formation of cell aggregates during starvation. The cells in these aggregates generate a pulse of the chemoattractant, cyclic adenosine 3’,5’-monophosphate (cAMP), every 6 min to 10 min, resulting in surrounding cells moving toward the aggregate. In addition to periodic pulses of cAMP, the cells also secrete phosphodiesterase (PDE), which degrades cAMP and prevents the accumulation of the chemoattractant. Here we show that small aggregates of Dictyostelium can disperse, with cells moving away from instead of toward the aggregate. This surprising behavior often exhibited oscillatory cycles of motion toward and away from the aggregate. Furthermore, the onset of outward cell motion was associated with a doubling of the cAMP signaling period. Computational modeling suggests that this dispersal arises from a competition between secreted cAMP and PDE, creating a cAMP gradient that is directed away from the aggregate, resulting in outward cell motion. The model was able to predict the effect of PDE inhibition as well as global addition of exogenous PDE, and these predictions were subsequently verified in experiments. These results suggest that localized degradation of a chemoattractant is a mechanism for morphogenesis.

A Perspective on Natural and Nature-Inspired Small Molecules Targeting Phosphodiesterase 9 (PDE9): Chances and Challenges against Neurodegeneration

As life expectancy increases, dementia affects a growing number of people worldwide. Besides current treatments, phosphodiesterase 9 (PDE9) represents an alternative target for developing innovative small molecules to contrast neurodegeneration. PDE inhibition promotes neurotransmitter release, amelioration of microvascular dysfunction, and neuronal plasticity. This review will provide an update on natural and nature-inspired PDE9 inhibitors, with a focus on the structural features of PDE9 that encourage the development of isoform-selective ligands. The expression in the brain, the presence within its structure of a peculiar accessory pocket, the asymmetry between the two subunits composing the protein dimer, and the selectivity towards chiral species make PDE9 a suitable target to develop specific inhibitors. Additionally, the world of natural compounds is an ideal source for identifying novel, possibly asymmetric, scaffolds, and xanthines, flavonoids, neolignans, and their derivatives are currently being studied. In this review, the available literature data were interpreted and clarified, from a structural point of view, taking advantage of molecular modeling: 3D structures of ligand-target complexes were retrieved, or built, and discussed.

Abstract P098: Statins Relax Systemic Mesenteric Arteries Via The Inhibition Of Phosphodiesterases

Purpose: Statins are the world’s most prescribed drugs with a range of vascular effects. Conventional view is that the vascular effects of statins are lipid-lowering-dependent and/or -independent, and both require long-term treatment. However, studies to examine direct vascular effects of statins are lacking as previous studies used supratherapeutic (≥1000-fold) concentrations of statins on non-resistance or cultured arteries that do not simulate physiology. Methods: Pressurized arterial myography, in-vitro phosphodiesterase (PDE) inhibition assay (colorimetric assay) and cGMP measurement using ELISA. Results: We found that, within 2-3 minutes of application, statins at 1nM produced mesenteric artery vasodilation that was reversed upon drug washout. Rosuvastatin and simvastatin produced vasodilation of ≥30μm while others dilated arteries by ≤20μm. Endothelium removal, or co-application of L-NAME, an inhibitor of endothelial nitric oxide (NO) synthase (eNOS), or ODQ, an inhibitor of smooth muscle cell guanylyl cyclase (sGC), or APC5, an inhibitor of protein kinase G (PKG), each inhibited statin-evoked vasodilation, suggesting the involvement of NO-sGC-PKG signaling axis. In contrast, mevalonate supplementation did not alter statin-induced vasodilation, precluding the role of lipid synthesis inhibition. To investigate potential involvement of phosphodiesterases (PDEs) that hydrolyze cGMP, we co-applied statins with a cGMP-specific PDE5 inhibitor sildenafil and found that statins indeed potentiated sildenafil-induced vasodilation. Our in-vitro PDE inhibition assay also showed that the vasodilatory statins have significant inhibitory actions on cGMP-dependent PDEs. Conclusion: Our study unveils a novel mechanism of systemic mesenteric artery vasodilation by statins. Future studies will examine if statins can be re-purposed for treating hypertension and/or erectile dysfunction.

Cigarette smoke exposure alters phosphodiesterases in human structural lung cells

Cigarette smoke (CS), a highly complex mixture containing more than 4,000 compounds, causes aberrant cell responses leading to tissue damage around the airways and alveoli, which underlies various lung diseases. Phosphodiesterases (PDEs) are a family of enzymes that hydrolyze cyclic nucleotides. PDE inhibition induces bronchodilation, reduces the activation and recruitment of inflammatory cells, and the release of various cytokines. Currently, the selective PDE4 inhibitor roflumilast is an approved add-on treatment for patients with severe chronic obstructive pulmonary disease with chronic bronchitis and a history of frequent exacerbations. Additional selective PDE inhibitors are being tested in preclinical and clinical studies. However, the effect of chronic CS exposure on the expression of PDEs is unknown. Using mRNA isolated from nasal and bronchial brushes and lung tissues of never smokers and current smokers, we compared the gene expression of 25 PDE coding genes. Additionally, the expression and distribution of PDE3A and PDE4D in human lung tissues was examined. This study reveals that chronic CS exposure modulates the expression of various PDE members. Thus, CS exposure may change the levels of intracellular cyclic nucleotides and thereby impact the efficiency of PDE-targeted therapies.